ES2742218T3 - Synthetic peptidoglycans that bind to collagen intended for use in vascular operations - Google Patents

Abstract

A collagen-binding peptidoglycan of formula (PnL) xG, where n is from 1 to 7, x is from 1 to 10, P is a synthetic peptide of up to 40 amino acids comprising a sequence of RRANAALKAGELYKSILY, L is a linker , and G is glycan, for use in a method of treating: i) a vascular injury; or ii) intimal hyperplasia.

Description

DESCRIPTION

Synthetic peptidoglycans that bind to collagen intended to be used in vascular operations TECHNICAL FIELD

[0001] This invention relates to the field of synthetic collagen binding peptidoglycans. More particularly, this invention relates to synthetic collagen-binding peptidoglycans for use in vascular intervention procedures.

BACKGROUND AND SUMMARY OF THE INVENTION

[0002] Vascular interventions involving a medical device inserted or implanted in a patient's body, for example, angioplasty, stent placement, atherectomy and grafting, are often associated with undesirable effects. For example, the insertion or implantation of catheters or stents can lead to the formation of emboli or clots in the blood vessels. Other adverse reactions to vascular intervention may include hyperplasia, restenosis, occlusion of blood vessels, platelet aggregation and calcification.

[0003] The number of percutaneous coronary intervention (PCI) procedures, commonly known as balloon angioplasty, has increased by 30% over the past 10 years for a total of more than 1.3 million patients in the US. . annually at a cost of more than $ 60 billion. During percutaneous coronary intervention, guide catheters move from the periphery, usually the femoral artery, to the aorta. The tip of the catheter is placed in the ostium of a coronary artery. Subsequently, cables, balloon catheters or other devices are advanced through the guiding catheter into the large epicardial coronary arteries to treat stenotic lesions.

[0004] The large increase in the number of procedures is due to an increase in heart disease, as well as technological advances, which has led to safer and more effective practice. However, PCI procedures are not without problems, such as thrombosis and intimal hyperplasia, which are complications of the procedure. The areas of focus to mitigate these complications are coagulation and inflammatory responses that occur in the vessel wall as a result of the procedure. Balloon inflation results in an endothelial denudation of the vascular wall, which initiates coagulation and inflammation through platelet activation and is currently a possible consequence of PCI procedures.

[0005] WO2009 / 120995A2 relates to synthetic collagen-binding peptidoglycans and collagen matrices designed comprising a collagen matrix and a synthetic collagen-binding peptidoglycan in which the synthetic collagen-binding pepididlycan is found.

[0006] Nili et al., Decorin Inhibition of PDGF-stimulated vascular smooth muscle cell function ", The American Journal of Pathology, September 2003, pages 869-878.

[0007] Paderi et al. "Collagen-Binding Peptidoglycans: A Biomimetic Approach to Modulate Collagen Fibrillogenesis for Tissue Engineering Applications", Tissue Engineering Part A, 2009, vol. 15, No. 10, pages 2991-2999.

[0008] Paderi et al. "Design of a Synthetic Collagen-Binding Peptidoglycan that Modulates Collagen Fibrillogenesis", Biomacromolecules, 2008, vol. 9, pages 2562-2566.

[0009] Lee et al. "Injectable gel with synthetic collagen-binding peptide for enhanced osteogenesis in vitro and in vivo", Biochemical and Biophysical Research Communications, Academic Press Inc., May 2007, vol. 357, No. 1, pages 68-74.

[0010] The synthetic collagen binding peptidoglycans described herein can be synthesized with design control and in large quantities at low cost, making their clinical use feasible. As described herein, synthetic collagen-binding pepidoglycans are designed to bind collagen with high affinity, where they remain attached during blood flow to prevent platelet binding to the exposed collagen of the naked endothelium and, consequently, to prevent platelet activation, thrombosis, inflammation as a result of undressing the endothelium, intimal hyperplasia and vasospasm. The synthetic collagen-binding peptidoglycans described herein can also stimulate proliferation of endothelial cells and can bind to collagen in a denuded vessel.

[0011] The present invention relates to a collagen binding peptidoglycan of formula (PnL) xG, for use in a method for the treatment of i) vascular injury or ii) intimal hyperplasia according to any one of claims 1 to 9.

[0012] The following numbered embodiments are contemplated:

1. A collagen binding peptidoglycan according to any one of claims 1 to 9 for use in vascular intervention in a patient, wherein the synthetic collagen binding peptidoglycan binds to a naked vessel in the patient.

2. The collagen binding peptidoglycan for use according to embodiment 1 wherein the synthetic collagen binding peptidoglycan inhibits platelet activation.

3. The collagen binding peptidoglycan for use according to any of embodiments 1 to 2, wherein the synthetic collagen binding peptidoglycan inhibits platelet binding to the denuded vessel. 4. The collagen binding peptidoglycan for use according to any of embodiments 1 to 3, wherein the synthetic collagen binding peptidoglycan inhibits intimal hyperplasia.

5. Collagen binding peptidoglycan for use according to any of embodiments 1 to 4, wherein the synthetic collagen binding peptidoglycan inhibits inflammation resulting from vessel stripping.

6. The collagen binding peptidoglycan for use according to any of embodiments 1 to 5, wherein the synthetic collagen binding peptidoglycan inhibits thrombosis.

7. Collagen binding peptidoglycan for use according to any of embodiments 1 to 6, wherein the synthetic collagen binding peptidoglycan inhibits vasospasm.

8. The collagen binding peptidoglycan for use according to any of embodiments 1 to 7, wherein the synthetic collagen binding peptidoglycan stimulates the proliferation of endothelial cells. 9. The collagen binding peptidoglycan for use according to any of embodiments 1 to 8, wherein the synthetic collagen binding peptidoglycan binds to the exposed collagen in the denuded vessel.

10. Collagen binding peptidoglycan for use according to any of embodiments 1 to 9, wherein the glycan is a glycosaminoglycan or a polysaccharide.

11. The collagen binding peptidoglycan for use according to any of embodiments 1 to 10, wherein the synthetic collagen binding peptidoglycan is DS-SILY-is.

12. Collagen binding peptidoglycan for use according to any of embodiments 1 to 11, wherein the synthetic collagen binding peptidoglycan is administered to the patient parenterally.

13. The collagen binding peptidoglycan for use according to embodiment 12 in which parenteral administration is through a route selected from the group consisting of intravascular, intravenous, intraarterial, intramuscular, cutaneous, subcutaneous, percutaneous, intradermal and intraepidermal

14. The collagen binding peptidoglycan for use according to any of embodiments 12 or 13 wherein the synthetic collagen binding peptidoglycan is administered parenterally using a needle or an infusion device.

15. Collagen-binding peptidoglycan for use according to any of embodiments 1 to 14, wherein the synthetic collagen-binding peptidoglycan is administered to the patient with a catheter, such as a balloon coating, through a porous balloon, or as a coating on a stent.

16. A kit that includes

a synthetic collagen-binding peptidoglycan for use according to claims 1 to 9; and a component selected from the group consisting of a catheter, a stent, a balloon and a combination thereof.

BRIEF DESCRIPTION OF THE DRAWINGS

[0013]

FIGURE 1 shows a schematic representation of the interaction between neighboring proteoglycans in adjacent tropocollagen chains that is important in determining the mechanical and alignment properties of collagen medications.

FIGURE 2. AFM images made in contact mode, with a scan rate of 2 Hz with silicon nitride contact mode tip k = tips of 0.05N / m and deviation setpoint: 0-1 volts, of gel samples prepared as in EXAMPLE 15 (10: 1 collagen: treatment) after dehydration with ethanol. The samples are for collagen alone (collagen) and for collagen with dermatan sulfate (DS), with decorin (Decorin), dermatan sulfate conjugate-RRANAALKAGELYKSILYGC (DS-SILY) and sulfao-SYIRIADTNIT (DS-SYIR) dermatan conjugate .

FIGURE 3. Surface plasmon resonance examination in association mode and dissociation mode of the RRANAALKAGELYKSILYGC (SILY) peptide that binds to the collagen bound to CM-3 plates. SILY was dissolved in 1x HBS-EP buffer at varying concentrations of 100 pm to 1.5 pm in 2-fold dilutions. FIGURE 4. Union of the modified SILY peptide with dansyl to the collagen measured in a 96-well high junction plate (black with a transparent bottom (Costar)). PBS, buffer only; BSA, BSA treated well; Collagen, well treated with collagen. Fluorescence readings were taken on an M5 Spectramax spectrophotometer (Molecular Devices) at excitation / emission wavelengths of 335nm / 490nm, respectively. FIGURE 5. Binding curve of the modified dansyl SILY peptide from the fluorescence data described in FIGURE 4.

FIGURE 6. A schematic description of the reagent, PDPH and the chemistry of the two-stage conjugation of a cysteine-containing peptide. with an oxidized glycosylaminoglycoside showing the release of 2-pyridylthiol in the final stage.

FIGURE 7. Union of the modified SILY peptide with dansyl conjugated with dermatan sulfate as described herein to the collagen measured in a 96-well high junction plate (black with a transparent bottom (Costar)). PBS, buffer only; BSA, well treated with BSA; Collagen, well treated with collagen. Fluorescence readings were taken on an Spectramax M5 spectrophotometer (Molecular Devices) at excitation / emission wavelengths of 335 nm / 490 nm respectively.

FIGURE 8. Measurement of the cutting module of the gel samples (1.5mg / ml of collagen III, collagen 5: 1: treatment) on an AR-G2 rheometer with a parallel stainless steel plate geometry of 20 mm (TA Instruments, New Castle, DE), and the geometry of the 20mm stainless steel parallel plate was reduced to a distance of 500 pm using a normal force control of 0.25N. ♦ - without treatment, that is, collagen III alone; ■ - dermatan sulfate collagen (1: 1); - dermatan sulfate collagen (5: 1); x - collagen sulfate-KELNLVYTGC (DS-KELN) conjugate (1: 1); A - collagen sulfate-KELN conjugate (5: 1); • - KELNLVYTGC peptide collagen (KELN).

FIGURE 9. Measurement of the cutting module of the gel samples (1.5mg / ml of collagen III, collagen 5: 1: treatment) on an AR-G2 rheometer with a parallel stainless steel plate geometry of 20 mm (TA Instruments, New Castle, DE), and the geometry of the 20mm stainless steel parallel plate was reduced to a separation distance of 500 pm using a normal force control of 0.25N. ♦ - without treatment, that is, collagen III alone; ■ - dermatan sulfate collagen (1: 1); - dermatan sulfate collagen (5: 1); x - collagen sulfate-GSIT conjugate (DS-GSIT) (1: 1); a - dermatan collagen sulfate - GSIT conjugate (5: 1); • - GSITTIDVPWNVGC (GSIT) peptide collagen.

FIGURE 10. Turbidity measurement. Gel solutions were prepared as described in EXAMPLE 15 (collagen 4 mg / ml and 10: 1 collagen for treatment, unless otherwise indicated) and 50 ml / well was added at 4 ° C at a 384 well plate. The plate was kept at 4 ° C for 4 hours before starting the formation of fibrils. A SpectraMax M5 at 37 ° C was used to measure the absorbance at 313 nm at 30 s intervals for 6 hours. Cabbage, without treatment, that is, collagen alone; DS, dermatan sulfate collagen; decorin, decorin collagen; DS-SILY, collagen sulfate conjugate from dermatan-SILY.

FIGURE 11. Cryoprint scanning electron microscopy images of the gel structure with an increase of 5000. Gels for cryo-SEM were formed, as described in EXAMPLE 18 (1 mg / ml collagen (Type III), collagen 1: 1: treatment), directly in the SEM stage. Images of regions with similar orientation were taken for comparison between treatments. Panel a, collagen, without treatment, that is, collagen alone; Panel b, dermatan sulfate collagen; Panel c, collagen sulfate-KELN conjugate; Panel d, collagen sulfate-GSIT conjugate.

FIGURE 12. The average empty space fraction measured from the Crio-SEM images shown in Figure 11. a) Collagen, without treatment, that is, only collagen; b) dermatan sulfate collagen; c) collagen sulfate-KELN conjugate; d) collagen sulfate-GSIT conjugate. All differences are significant with p = 0.05.

FIGURE 13. Measurement of absorbance at 343 nm before treatment of oxidized heparin conjugated to PDPH, and after treatment with SILY, which releases 2-pyridylthiol from the conjugate and allows the determination of the proportion of SILY peptide conjugated to oxidized heparin. The measured AA corresponds to 5.44 SILY molecules / oxidized heparin.

FIGURE 14. Characterization of the DS-SILY conjugation. After 2 hours, a final AA343nm corresponded to 1.06 SILY molecules added to each DS molecule. Note that t = 0 is an approximate time point of zero due to the slight delay between adding SILY to the DS-PDPH and measuring the solution at 343 nm.

FIGURE 15. Conjugation of Dcl3 to DS. The production of pyridine-2-thione measured by an increase in absorbance at 343 nm indicates 0.99 Dc13 peptides per DS polymer chain.

FIGURE 16. Microplate fluorescence binding of DS-ZDc13 to collagen. DS-ZDc13 specifically bound to the surface of the collagen in a dose-dependent manner.

FIGURE 17. Collagen fibrinogenesis by turbidity measurements. DS-Dc13 delays fibrinogenesis and decreases overall absorbance in a dose-dependent manner. The free Dc13 peptide, on the other hand, seems to have little effect on fibrinogenesis compared to collagen only in the high molar ratio of collagen: additive of 1: 1.

FIGURE 18. Average diameter of Crio-SEM fibrils. A. Decorin and synthetic peptidoglycans significantly decrease the diameter of the fibrils on the collagen or the collagen DS. B. Compared to collagen alone, the free peptide Dc13 does not affect the diameter of the fibrils, while SILY causes a decrease in the diameter of the fibrils.

FIGURE 19. Gel compaction. A. and B. Days 3 and 5, respectively: decorin and peptidoglycans are significant in relation to collagen and DS, * indicates that DS-Dc13 and DS are not significant on day 3. Bars do not indicate no meaning C. Day 7: Decorin is significant against all samples, n ° DS is significant compared to collagen. D. Day 10: + Collagen and DS are significant, $ DS-Dc13 is significant compared to decorin and collagen.

FIGURE 20. Estimate elastin by Fastin test. A. DS-SILY significantly increased elastin production in all samples. DS and DS-Dc13 significantly decreased elastin production on collagen. Control samples of collagen gels without cells showed no elastin production. B. Free peptides resulted in a slight decrease in elastin production compared to collagen, but no point was significant.

FIGURE 21. SEM images of slides incubated with platelet rich plasma. Arrows in heparin-SILY treatment indicate structures similar to the unique fibrils of this treatment. Scale bar = 100 pm.

FIGURE 22. Density of Crio-SEM fibrils. Density of fibrils, defined as the relationship between the area that contains the fibrils and the empty space. DS-SILY and the free SILY peptide had a significantly higher fibril density, while the collagen had a significantly lower fibril density. DS-Dc13 was not significant compared to collagen.

FIGURE 23. Storage module (G ') of collagen gels. Rheological mechanical tests of collagen gels formed with each additive at A. 5: 1 B. 10: 1 and C. 30: 1 molar ratio of collagen: additive. Frequency sweeps of 0.1 Hz to 1.0 Hz were performed with a controlled voltage of 1.0 Pa. G'avg ± S.E.

FIGURE 24. Cell proliferation and cytotoxicity assays. No significant differences were found among all additives in A. CyQuant B. Live tests and C. Dead.

FIGURE 25. Cryo-SEM images for fibril density. Collagen gels formed in the presence of each additive at 10: 1 molar ratio of collagen: additive. A. DS, Decorin, or peptidoglycans. B. Free peptides. The images are taken at 10,000x, scale bar = 5 pm.

FIGURE 26. AFM images of collagen gels. Collagen gels were formed in the presence of each additive in a 10: 1 molar ratio of collagen: additive. D band is observed for all additives. The images are 1 pm2

FIGURE 27. Inhibition of platelet activation. Measurement determining the release of activation factors Platelet factor 4 (PF-4) and p-thromboglobulin (Nap-2). The collagen immobilized on the surface of a 96-well plate was previously incubated with each treatment and subsequently incubated with platelet rich plasma (PRP). Values are reported as a percentage of the activation factor released by the treatment compared to the amount of activation factor released by the control treatment (phosphate buffered saline, PBS). The * indicates that the difference is significant compared to the untreated collagen surface (phosphate buffered saline, PBS). Dex, dextran; Dex-SILY9, dextran conjugate- (SILY) g; Hep, heparin; Hep-SILY, heparin-SILY conjugate; HA, hyaluronan; HA-SILY, hyaluronano-SILY conjugate; SILY, SILY peptide. Due to the solubility limits, Hep, Hep-SILY, HA and HA-SILY were incubated at 25 pm. All other treatments were performed at 50 pm (once the treatment was removed, the plates were washed with PBS <1 min, before the addition of PRP). The conjugates of Hep and HA (hyaluronic acid) contained approximately 4 peptides per polysaccharide.

FIGURE 28. Inhibition of platelet activation. Measurement determining the release of activation factors Platelet factor 4 (PF-4) and p-thromboglobulin (Nap-2). The collagen immobilized on the surface of a 96-well plate was previously incubated with each treatment and subsequently incubated with platelet rich plasma (PRP). Values are reported as a percentage of the activation factor released by the treatment compared to the amount of activation factor released by the control treatment (phosphate buffered saline, PBS). Dex, dextran; Dex-SILY6, dextran conjugate- (SILY) 6; Hep, heparin; Hep-GSIT, heparin-GSIT conjugate; GSIT, GSIT peptide; SILY, SILY peptide. The measured values for all treatments are significant against ^p B ^s . Dex, SILY and Dex-SILY6 are at 25 pm, all other treatments are at 50 pm. The ** indicates that the value for the treatment with Hep-GSIT was significant compared to the values for the treatment of Hep, similarly the value for the treatment with Dex-SILY6 was significant compared to the value for the treatment with Dex for PF4. (After the treatment was removed, the plates were rinsed for 20 minutes). Hep conjugates contained approximately 4 peptides per polysaccharide.

FIGURE 29. Inhibition of platelet binding to collagen by a colorimetric assay. The collagen immobilized on the surface of a 96-well plate was previously incubated with each treatment and subsequently incubated with platelet rich plasma (PRP). The microplate assay prepared as described was pre-incubated with collagen treatments, only PBS; Dextran; Dex-SILY6, dextran- (SILY) 6; SILY, SILY peptide. * Significant vs. collagen (without treatment).

FIGURE 30. Fluorescence image of adhered platelets. The adhered platelets were fixed with 4% paraformaldehyde, permeabilized with 0.1% Triton X-100 and the platelet actin was labeled with faloidin-AlexaFluor 488. Images of the adhered platelets were taken using a vertical fluorescence microscope using a vertical fluorescence microscope using DAPI filter Without treatment, that is, PBS treated collagen.

FIGURE 31. Fluorescence image of adhered platelets. The adhered platelets were fixed with 4% paraformaldehyde, permeabilized with 0.1% Triton X-100 and the platelet actin was labeled with faloidin-AlexaFluor 488. Images of the adhered platelets were taken using a vertical fluorescence microscope using a vertical fluorescence microscope using DAPI filter Treatment: dextran.

FIGURE 32. Fluorescence image of adhered platelets. The adhered platelets were fixed with 4% paraformaldehyde, permeabilized with 0.1% Triton X-100 and the platelet actin was labeled with faloidin-AlexaFluor 488. Images of the adhered platelets were taken using a vertical fluorescence microscope using a vertical fluorescence microscope using DAPI filter Treatment: dextran-SILY9 conjugate.

FIGURE 33. Fluorescence image of adhered platelets. The adhered platelets were fixed with 4% paraformaldehyde, permeabilized with 0.1% Triton X-100 and the platelet actin was labeled with faloidin-AlexaFluor 488. Images of the adhered platelets were taken using a vertical fluorescence microscope using a vertical fluorescence microscope using DAPI filter Treatment: hyaluronan. Figure 34. Fluorescence image of adhered platelets. The adhered platelets were fixed with 4% paraformaldehyde, permeabilized with 0.1% Triton X-100 and the platelet actin was labeled with faloidin-AlexaFluor 488. Images of the adhered platelets were taken using a vertical fluorescence microscope using a vertical fluorescence microscope using DAPI filter Treatment: Hyaluron-SILY conjugate.

FIGURE 35. Fluorescence image of adhered platelets. The adhered platelets were fixed with 4% paraformaldehyde, permeabilized with 0.1% Triton X-100 and the platelet actin was labeled with faloidin-AlexaFluor 488. Images of the adhered platelets were taken using a vertical fluorescence microscope using a vertical fluorescence microscope using DAPI filter Treatment: heparin. FIGURE 36. Fluorescence image of adhered platelets. The adhered platelets were fixed with 4% paraformaldehyde, permeabilized with 0.1% Triton X-100 and the platelet actin was labeled with faloidin-AlexaFluor 488. Images of the adhered platelets were taken using a vertical fluorescence microscope using a vertical fluorescence microscope using DAPI filter Treatment: heparin-SILY conjugate.

FIGURE 37. Fluorescence image of adhered platelets. The adhered platelets were fixed with 4% paraformaldehyde, permeabilized with 0.1% Triton X-100 and the platelet actin was labeled with faloidin-AlexaFluor 488. Images of the adhered platelets were taken using a vertical fluorescence microscope using a vertical fluorescence microscope using DAPI filter Treatment: SILY peptide.

FIGURE 38. Collagen degradation determined by hydroxyproline. Treatments: Ctrl, without added cells; Cabbage, collagen without further treatment; DS, dermatan sulfate; Decorin; DS-SILY, dermatan sulfate-SILY conjugate; DS-Dc13, dermatan sulfate conjugate Dc13; SILY, SILY peptide; Dcl3, Dc13 peptide.

FIGURE 39. Inhibition of platelet activation. Measurement determining the release of the activation factors Platelet Factor 4 (PF-4) and p-thromboglobulin (Nap-2). Type I and III collagen gels on the surface of a 96-well plate were pre-incubated with each treatment and subsequently incubated with PRP. Platelet activation was measured by the release of the activation factors PF-4 and Nap-2. Treatments: PBS, buffer alone; Dex, dextran; Dex-SILY, dextran-SILY conjugate; Dex-GSIT, dextran-GSIT conjugate; Dex-KELN, dextran-KELN conjugate; Dex-Dcl3, dextran-Dcl3 conjugate; SILY, SILY peptide; GSIT, GSIT peptide; KELN, KELN peptide; Dc13, Dc13 peptide; Dex-SILY Dex-GSIT; combination of dextran-SILY conjugate and dextran-GSIT conjugate; SILY GSIT; Combination of SILY peptide and GSIT peptide. * Indicates that the results are significant compared to the untreated collagen surface (PBS). ** Indicates that the results are also significant compared to the collagen surface with Dex. *** Indicates that the results are also significant compared to the collagen surface with the corresponding peptide control. All peptidoglycans caused a significant decrease in NAP-2 release compared to no treatment (PBS) or dextran treatment, while Dex-GSIT also decreased release over its peptide control (GSIT). Dex-GSIT and Dex-KELN significantly decreased the release of PF-4 in relation to no treatment (PBS) and treatment with dextran, while Dex-Dc13 significantly decreased the release of PF-4 over no treatment (PBS).

FIGURE 40. Inhibition of platelet binding to collagen (adhesion) by a colorimetric assay. Treatments: PBS, buffer alone; Dex, dextran; Dex-SILY, dextran-SILY conjugate; Dex-GSIT, dextran-GSIT conjugate; Dex-KELN, dextran-KELN conjugate; Dex-Dc13, dextran-Dc13 conjugate; SILY, SILY peptide; GSIT, GSIT peptide; KELN, KELN peptide; Dc13, Dc13 peptide; Dex-SILY Dex-GSIT; combination of dextran-SILY conjugate and dextran-GSIT conjugate; SILY GSIT; Combination of SILY peptide and GSIT peptide. * Significant surface against untreated collagen (PBS). ** Also significant against the surface of collagen with Dex. *** Also significant against the collagen surface with the corresponding peptide control. Dex-SILY and Dex-KELN had a significant decrease in platelet adhesion compared to no treatment (PBS) or treatment with Dextran, while Dex-GSIT also decreased platelet adhesion on their peptide control treatment ( GSIT).

FIGURE 41 shows the purification of DS-BMPH. The number of BMPH crosslinkers attached to DS is determined by calculating the excess BMPH that is then subtracted from the known amount added, which produces the amount that reacts with the oxidized DS. The excellent separation of the two molecular species is achieved under purification procedures, which is demonstrated by the wide separation of the peaks. FIGURE 42 shows the oxidation of the periodate. As the amount of sodium meta-periodate increases during DS oxidation, the number of BMPH crosslinkers per DS chain increases linearly.

FIGURE 43 shows the affinity for peptidoglycan binding. The biotin labeled peptidoglycans DS-SILY4 and DS-SILY18 were synthesized and incubated on a surface of fibrillar collagen. After washing, bound peptidoglycan was detected and saturation binding curves were adjusted to calculate binding affinities. DS-SILY4 and DS-SILY18 bind to collagen with K ^d = 118 nM and 24 nM respectively, which shows that increasing the number of peptides increases the affinity of peptidoglycan to collagen. In addition, a greater number of peptides increases the amount of peptidoglycan that binds to the surface, which is observed by the increase in absorbance. Note DS-SILY18 does not contain more biotin-labeled peptides than DS-SILY4.

FIGURE 44 shows the percentage decrease in the release of PF4 and NAP2 activation factors compared to untreated collagen surfaces (NT). DS, SILY or DS-SILY were incubated for 15 minutes at a concentration of 50 µm and then rinsed off the collagen surface for 24 hours. * indicates meaning for NT, ** indicates meaning for NT, and DS, and SILY a = 0.05.

FIGURE 45 shows the inhibition of platelet activation. Fibrillar collagen surfaces were incubated with varying concentrations of DS-SILY18 peptidoglycan. Unbound peptidoglycan was rinsed from the surface for 24 hours. Human platelets were incubated on the surface and activation was measured by the release of PF-4 and Nap-2 following FDA guidelines. Maximum inhibition of platelet activation was achieved in 10 jm concentrations.

FIGURE 46 shows the diffusion of DS-SILY18 from a fibrillar collagen surface. The labeled DS-SILY18 was attached to a collagen surface as described and incubated at 37 ° C with an extensive rinse for up to 11 days. The detection of peptidoglycan at several time points showed that it diffuses from the surface over time, but even after 1 week, the equivalent of approximately 10 nM remained attached.

FIGURE 47 shows the proliferation of endothelial cells. Proliferation was measured in the presence of varying concentrations of peptidoglycan to determine if peptidoglycan had an adverse effect on endothelial regrowth. At the highest concentration tested, a significant increase in cell proliferation was observed. * indicates significance a = 0.05, n = 6, presented as standard average. dev. FIGURE 48 shows endothelial migration on treated collagen surfaces. To more closely mimic the environment of a bare vessel with exposed collagen, endothelial growth in collagen with varying concentrations of bound peptidoglycan was analyzed. At higher concentrations of peptidoglycans there was a significant increase in endothelial cell migration. * indicates significance a = 0.05, n = 6, presented as average standard development.

FIGURE 49 shows the binding of platelets to collagen under flow. Platelet rich human plasma was analyzed under flow for platelet fixation on fibrillar collagen surfaces. Treatment conditions Collagen surfaces treated with DS-SILY (Panel A) or untreated (Panel B) show significantly fewer platelets attached to the surface treated with peptidoglycan.

FIGURE 50 shows a schematic representation of the inhibition by peptidoglycan of platelet binding and activation in the collagen of the denoted endothelium.

FIGURE 51 shows the quantification of platelet junction inhibited by vasospasm. Panel A shows a representative angiography profile of treated and untreated balloon injured vessels. Vasospasm is evident in the untreated vessel, while treatment with peptidoglycan does not have vasospasm. Panel B shows quantified vasospasm by measuring the% occlusion of the vessel using angiography data. A total of 12 ball injuries, 7 untreated and 5 treated, were analyzed. * indicates importance compared to p = 0.005 untreated.

FIGURE 52 shows the histological evaluation of the injured vessels by balloon using Verhoff-Van Gieson staining. Intimate hyperplasia is evident in the simulated control (panel A) as seen by the growth of the internal elastic sheet. In vessels treated with peptidoglycan, intimal hyperplasia is absent (panel B).

FIGURE 53 shows denuded arteries incubated with Ix PBS (Panel A: Control) or labeled peptidoglycan (Panel B: Peptidoglycan (10 jm DS-SILY18-biotin)).

FIGURE 54 shows the inhibition by DS-SILY18 of the binding of whole blood to collagen under flow.

DETAILED DESCRIPTION OF THE ILLUSTRATIVE EMBODIMENTS

[0014] As used in accordance with this invention, a "synthetic collagen-binding peptidoglycan" means a conjugate of a binding glycan with a synthetic collagen peptide. "Collagen-binding synthetic peptidoglycans" may have an amino acid homology with a portion of a protein or a proteoglycan that normally does not participate in collagen fibrinogenesis or may have an amino acid homology with a portion of a protein or a proteoglycan that normally participates in the fibrinogenesis of collagen.

[0015] These synthetic collagen binding proteoglycans can be used in vascular intervention procedures including, for example, to prevent any one or a combination of platelets from binding to exposed collagen of the denoted endothelium, platelet activation, thrombosis, resulting inflammation. of denotation of the endothelium, intimal hyperplasia, and vasospasm. The synthetic collagen-binding peptidoglycans described herein can also stimulate proliferation of endothelial cells and can bind collagen in a denuded vessel.

[0016] In various embodiments described herein, the synthetic collagen binding peptidoglycans described comprise synthetic peptides of about 5 to about 40 amino acids. In some embodiments, these peptides have homology to the amino acid sequence of a small leucine-rich proteoglycan, a platelet receptor sequence or a protein that regulates collagen fibrillogenesis. The synthetic peptide is an amino acid sequence of RRANAALKAGELYKSILY and an amino acid sequence with a homology of 80%, 85%, 90%, 95% or 98% with the amino acid sequence. In a disclosure that is not within the scope of the invention, the synthetic peptide may also be any peptide of 5 to 40 amino acids selected from peptides that have collagen binding activity and that are 80%, 85%, 90%, 95% , 98%, or 100% homologous with the collagen binding domain (s) of von Willebrand factor or a platelet collagen receptor as described in Chiang, et al., J. Biol. Chem. 277: 34896-34901 (2002), Huizinga, et al., Structure 5: 1147-1156 (1997), Romijn, et al., J Biol. Chem. 278: 15035-15039 (2003), and Chiang, et al., Cardio. & Haemato. Disorders-Durg Targets 7: 71-75 (2007).

[0017] Glucan (for example glycosaminoglycans, abbreviated GAG, or polysaccharide) bound to the synthetic peptide may be selected from the group consisting of alginate, agarose, dextran, chondroitin, dermatane, dermatan sulfate, heparan, heparin, keratin, and Hyaluronan In one embodiment, the glycan is selected from the group consisting of dermatan sulfate, dextran and heparin. In another illustrative embodiment, the glycan is dermatan sulfate. The synthetic collagen-binding proteoglycan in any of these embodiments can be used to inhibit platelet binding to the exposed collagen of the denoted endothelium, platelet activation, thrombosis, inflammation resulting from undressing the endothelium, intimal hyperplasia and the vasospasm during a vascular intervention procedure. The synthetic collagen-binding peptidoglycans described herein can also stimulate proliferation of endothelial cells and can bind collagen in a denuded vessel.

[0018] In an illustrative aspect, the synthetic collagen-binding peptidoglycan can be sterilized. As used herein, "sterilization" or "sterilization" or "sterilization" means disinfecting synthetic peptidoglycans that bind to collagen by removing unwanted contaminants, including endotoxins and infectious agents.

[0019] In various illustrative embodiments, the synthetic collagen-binding peptidoglycan can be disinfected and / or sterilized using conventional sterilization techniques including propylene oxide or treatment with ethylene oxide, gas plasma sterilization, gamma radiation, beam of electrons, and / or sterilization with a peracid, such as peracetic acid.

Sterilization techniques that do not adversely affect the structure and biotropic properties of the synthetic peptidoglycan that binds to collagen can be used. Illustrative sterilization techniques are exposure of synthetic collagen-binding peptidoglycan to peracetic acid, to gamma irradiation of 1-4 Mrads (or 1-2.5 Mrads of gamma irradiation), to treatment with ethylene oxide, to sterile filtration or plasma gas sterilization. In one embodiment, the synthetic collagen-binding peptidoglycan can be subjected to one or more sterilization processes. Another illustrative embodiment is to subject the synthetic collagen binding proteoglycan to sterile filtration. The synthetic collagen-binding peptidoglycan can be wrapped in any type of container that includes a plastic wrap or an aluminum wrap, and can be further sterilized.

[0020] In various embodiments described herein, the binding collagen can be combined with synthetic peptidoglycan minerals, amino acids, sugars, peptides, proteins, vitamins (such as ascorbic acid), or laminin, collagen, fibronectin, hyaluronic acid, fibrin, elastin, or aggrecan, or growth factors such as epidermal growth factor, platelet-derived growth factor, beta transforming growth factor or fibroblast growth factor, and glucocorticoids such as dexamethasone or viscoelastic altering agents, such as ionic polymers and non-ionic water soluble .; acrylic acid polymers; hydrophilic polymers such as polyethylene oxides, polyoxyethylene-polyoxypropylene copolymers and polyvinyl alcohol; cellulosic polymers and cellulosic polymer derivatives such as hydroxypropylcellulose, hydroxyethylcellulose, hydroxypropylmethylcellulose, hydroxypropylmethylcellulose phthalate, methylcellulose, carboxymethylcellulose and etherified cellulose; polylactic acid, polyglycolic acid, copolymers of lactic and glycolic acids, or other polymeric agents, both natural and synthetic.

[0021] In various embodiments described herein, a kit is provided comprising one or more collagen-binding synthetic peptidoglycans. The kit itself may be inside a container of any type, and the kit may contain instructions for the use of the kit components. In one embodiment, the kit comprises a container, vial, container, bag or wrap, for example, which contains a synthetic peptidoglycan that binds to the collagen. In another embodiment, the kit comprises a separate container or containers (for example, a vial, container, bag or wrapper), each of which contains one of the following components: a buffer and one or more types of synthetic peptidoglycans that are bind to collagen. In any of these embodiments, the kits may further comprise a buffer, a sterilizing or disinfecting agent, non-collagen proteins or polysaccharides and / or instructional materials that describe methods for using the kit reagents. In any of these embodiments, the kit may contain a component selected from the group consisting of a catheter, a stent, a balloon and a combination thereof. The synthetic peptidoglycan that binds to collagen can be lyophilized, for example, in a buffer or in water.

[0022] In any of the embodiments described herein, the synthetic collagen-binding peptidoglycan can be a compound of the following formula

(PnL) xG in which n is from 1 to 7;

where x is 1 to 10;

wherein P is a synthetic peptide of about 5 to about 40 amino acids comprising a sequence of RRAn Aa LKAGELYKSILY; where L is a linker; Y

where G is a glycan.

[0023] In the formula described above, n can be from 1 to 5.

[0024] In various embodiments described herein, a synthetic collagen binding peptidoglycan comprising a synthetic peptide of about 5 to about 40 amino acids with amino acid sequence homology to a collagen binding peptide (eg, a portion of an amino acid sequence of a collagen or proteoglycan binding protein) conjugated to alginate, agarose, dextran, chondroitin, dermatan, dermatan sulfate, heparan, heparin, keratin and hyaluronan. In one embodiment, the glycan is selected from the group consisting of dermatan sulfate, dextran, hyaluronan and heparin. In another illustrative embodiment, the glycan is dermatan sulfate. In yet another embodiment, the glycan is dermatan sulfate with 18 peptides of the RRANAALKAGELYKSILYGC sequence linked to the glycan (ie, DS-SlLY-ia). In yet another embodiment, glycan is dermatan sulfate with 18 peptides comprising the RRANAALKAGELYKSILY sequence linked to glycan. The synthetic collagen binding proteoglycan in any of these embodiments can be used to inhibit platelet binding to the exposed collagen of the naked endothelium, inhibit the binding of other cells in the blood to the exposed collagen of the naked epithelium, inhibit platelet activation, inhibit thrombosis, inhibit inflammation as a result of undressing the endothelium, inhibit intimal hyperplasia and / or inhibit vasospasm. The synthetic collagen-binding peptidoglycans described herein can also stimulate proliferation of endothelial cells and can bind collagen in a denuded vessel. In any of these embodiments, these effects mentioned above may occur during a vascular intervention procedure, such as a catheter-based procedure. In any of these embodiments, any of the compounds described above can be used.

[0025] In another illustrative embodiment, any of the compounds described above as embodiments A, B, or C or alternative embodiments A or B can inhibit exposed collagen-binding platelets of the denoted endothelium, platelet activation, thrombosis, inflammation resulting from denudation the endothelium, intimal hyperplasia and / or vasospasm, or it can stimulate proliferation of endothelial cells or it can bind to collagen in a denuded vessel. In another illustrative embodiment, during a vascular intervention procedure, any of the compounds described above as embodiments A, B or C or alternative embodiments A or B, can inhibit platelet binding to the exposed collagen of the denoted endothelium, the activation of the Platelets, thrombosis, inflammation resulting from undressing the endothelium, intimal hyperplasia and / or vasospasm, or it can stimulate proliferation of endothelial cells or it can bind to collagen in a denuded vessel. In another illustrative embodiment, during a vascular intervention procedure, any of the compounds described above as embodiments A, B or C or alternative embodiments A or B, can inhibit platelet binding to the exposed collagen of the denoted endothelium, the activation of the Platelets, intimate hyperplasia and / or vasospasm, or can bind to collagen in a denuded vessel.

[0026] In another illustrative embodiment, DS-SlLY-ia can inhibit platelet binding to exposed collagen of the denoted endothelium, platelet activation, thrombosis, inflammation resulting from denotation of the endothelium, intimal hyperplasia, and / or vasospasm, or It can stimulate endothelial cell proliferation or it can bind to collagen in a denuded vessel. In another illustrative embodiment, during a vascular intervention procedure, DS-SlLY-ia can inhibit platelet binding to the exposed collagen of the denoted endothelium, platelet activation, thrombosis, inflammation resulting from denotation of the endothelium, intimal hyperplasia and / or vasospasm, or it can stimulate endothelial cell proliferation or it can bind to collagen in a denuded vessel. In another illustrative embodiment, during a vascular intervention procedure, DS-SILY18 can inhibit platelet binding to the exposed collagen of the denoted endothelium, activation of platelets, intimal hyperplasia and / or blood vessels, or it can bind to collagen in a denuded glass.

[0027] In various embodiments described herein, the synthetic peptides described herein can be modified by including one or more conservative amino acid substitutions. As is well known to those skilled in the art, the alteration of any non-critical amino acid of a peptide by a conservative substitution should not significantly alter the activity of that peptide because the side chain of the replacement amino acid should be able to form similar bonds and contacts with the side chain of the amino acid that has been replaced.

[0028] Non-conservative substitutions are possible, provided they do not excessively affect the collagen binding activity of the peptide and / or reduce its effectiveness in inhibiting platelet activation, platelet binding to exposed collagen of the denoted endothelium, activation of platelets, thrombosis, inflammation as a result of undressing the endothelium, intimal hyperplasia and / or vasospasm, or its effectiveness in stimulating the proliferation of endothelial cells or to bind to collagen in a denuded vessel.

[0029] As is well known in the art, a "conservative substitution" of an amino acid or a "conservative substitution variant" of a peptide refers to an amino acid substitution that maintains: 1) the secondary structure of the peptide; 2) the charge or hydrophobicity of the amino acid; and 3) the volume of the side chain or one or more of these characteristics. Illustratively, the well-known terminologies "hydrophilic residues" refer to serine or threonine. "Hydrophobic residues" refer to leucine, isoleucine, phenylalanine, valine or alanine. "Positively charged residues" refer to lysine, arginine, omitin or histidine. "Negatively charged residues" refer to aspartic acid or glutamic acid. Waste that has "bulky side chains" refers to phenylalanine, tryptophan or tyrosine. A list of illustrative conservative amino acid substitutions is provided in Table 1.

TABLE 1

[0030] In the various conservative amino acid substitution embodiments described herein, a synthetic collagen binding peptidoglycan comprising a synthetic peptide of about 5 to about 40 amino acids with the amino acid sequence homology to a collagen binding peptide (for example, a portion of an amino acid sequence of a collagen or proteoglycan binding protein) conjugated to a glycan selected from the group consisting of alginate, agarose, dextran, chondroitin, dermatan, dermatan sulfate, heparin, heparin, keratin , and hyaluronan can be used. In one embodiment, the glycan is selected from the group consisting of dermatan sulfate, dextran, hyaluronan and heparin. In another illustrative embodiment, the glycan is dermatan sulfate. In yet another embodiment, the glycan is dermatan sulfate with 18 peptides of the RRANAALKAGELYKSILYGC sequence linked to the glycan and this sequence can be conservatively substituted. The synthetic collagen binding proteoglycan in any of these conservative substitution embodiments can be used to inhibit platelet binding to the exposed collagen of the denoted endothelium, platelet activation, thrombosis, inflammation resulting from endothelial denudation, intimal hyperplasia and vasospasm. The synthetic collagen binding peptidoglycans described herein with conservative amino acid substitutions can also stimulate proliferation of endothelial cells and can bind to collagen in a denuded vessel. In any of these embodiments, these effects mentioned above may occur during a vascular intervention procedure, such as a catheter-based procedure. In any of these conservative substitution embodiments, any of the compounds described above can be used.

[0031] In another illustrative description, any of the compounds selected from the group consisting RRANAALKAGELYKSILYGC, RLDGNEIKRGC, AHEEISTTNEGVMGC, NGVFKYRPRYFLYKHAYFYPPLKRFPVQGC, CQDSETRTFY, TKKTLRTGC, GLRSKSKKFRRPDIQYPDATDEDITSHMGC, SQNPVQPGC, SYIRIADTNITGC, SYIRIADTNIT, KELNLVYT, KELNLVYTGC, GSITTIDVPWNV, GELYKSILYGC, GSITTIDVPWNVGC and GCGGELYKSILY that They have conservative amino acid substitutions can be used. In any of these embodiments, compounds with conservative amino acid substitutions can inhibit platelet binding to the exposed collagen of the denoted endothelium, platelet activation, thrombosis, inflammation resulting from undressing the endothelium, intimate hyperplasia 1 and / or vasospasm, or they can stimulate endothelial cells. proliferation or can bind to collagen in a denuded vessel. In another illustrative embodiment, during a vascular intervention procedure, any of these compounds with conservative amino acid substitutions can inhibit platelet binding to the exposed collagen of the denoted endothelium, platelet activation, thrombosis, inflammation resulting from denudation. of the endothelium, intimal hyperplasia and / or vasospasm, or it can stimulate proliferation of endothelial cells or it can bind to collagen in a bare vessel. In another illustrative embodiment, during a vascular intervention procedure, any of the compounds with conservative amino acid substitutions described in This paragraph may inhibit platelet binding to the exposed collagen of the denoted endothelium, activation of platelets, intimal hyperplasia and / or vasospasm, or collagen may be attached in a stripped container.

[0032] In various embodiments described herein, the synthetic peptide is synthesized according to solid phase peptide synthesis protocols that are well known to those skilled in the art. In one embodiment, a peptide precursor is synthesized on a solid support in accordance with the well-known Fmoc protocol, cleaved from the support with trifluoroacetic acid and purified by chromatography according to methods known to those skilled in the art.

[0033] In various embodiments described herein, the synthetic peptide is synthesized using biotechnology methods that are well known to persons skilled in the art. In one embodiment, a DNA sequence encoding the amino acid sequence information for the desired peptide is ligated by recombinant DNA techniques known to those skilled in the art in an expression plasmid (for example, a plasmid that incorporates a tag affinity for peptide affinity purification), the plasmid is transfected into a host organism for expression, and the peptide is then isolated from the host organism or growth medium according to the methods known to those skilled in the art ( for example, by affinity purification). The methods of recombinant DNA technology are described in Sambrook et al., "Molecular Cloning: A Laboratory Manual", 3rd edition, Cold Spring Harbor Laboratory Press, (2001), and are well known to those skilled in the art.

[0034] In various embodiments described herein, the synthetic peptide is conjugated to a glycan by reacting a free amino group of the peptide with an aldehyde function of the glycan in the presence of a reducing agent, using methods known to those skilled in the art. technique, to produce the conjugated glycan peptide. In one embodiment, an aldehyde function of glycan (for example, polysaccharide or glycosaminoglycan) is formed by reacting the glycan with sodium metaperiodate according to methods known to those skilled in the art.

[0035] In one embodiment, the synthetic peptide is conjugated to a glycan by reacting an aldehyde function of glycan with propionyl 3- (2-pyridyldithio) hydrazide (CPPD) to form an intermediate glycan and further reacting the intermediate glycan with a peptide containing a free thiol group to produce the conjugated glycan peptide. In yet another embodiment, the peptide sequence can be modified to include a glycinecysteine segment to provide a binding point for a glycan or a glucan linker conjugate.

[0036] In any of the embodiments described herein, the synthetic peptide is conjugated to a glycan by reacting an aldehyde function of glycan with a crosslinker, for example, 3- (2-pyridyldithio) propionyl hydrazide (CPPD), to form an intermediate glycan and further react the intermediate glycan with a peptide containing a free thiol group to produce the glycan peptide conjugate. In any of the various embodiments described herein, the peptide sequence may be modified to include a glycine-cysteine segment to provide a point of attachment for a glycan or a glucan-linker conjugate. In any of the embodiments described herein, the crosslinker may be N- [pmaleimidopropionic acid] hydrazide (BMPH).

[0037] Although the specific embodiments have been described in the preceding paragraphs, the synthetic collagen binding peptidoglycans described herein can be performed by using any method recognized in the art for conjugation of the peptide to glycan (eg, polysaccharide or glycosaminoglycan). This may include covalent, ionic or hydrogen bonds, either directly or indirectly through a linkage group, such as a divalent linker. The conjugate is typically formed by a covalent bond of the peptide to the glycan through the formation of the amide, ester or imino bonds between the acid, aldehyde, hydroxy, amino or hydrazine groups in the respective components of the conjugate. All these methods are known in the art or are described in more detail in the Examples section of this application or in Hermanson GT, Bioconjugate Techniques, Academic Press, pp. 169-186 (1996). The linker typically comprises from about 1 to about 30 carbon atoms, more typically from about 2 to about 20 carbon atoms. Typically, low molecular weight linkers are used (ie, those having an approximate molecular weight of about 20 to about 500).

[0038] In addition, structural modifications of the linker portion of the conjugates are contemplated herein. For example, amino acids can be included in the linker and several amino acid substitutions can be made to the linker portion of the conjugate, including natural amino acids, as well as those available from conventional synthetic methods. In another aspect, longer chain beta, gamma and amino acids may be used instead of one or more alpha amino acids. In another aspect, the linker can be shortened or lengthened, either by changing the number of amino acids included therein, or by including more or less beta, gamma or longer chain amino acids. Similarly, the length and shape of other chemical fragments of the linkers described herein can be modified.

[0039] In various embodiments described herein, the linker may include one or more bivalent fragments independently selected in each case from the group consisting of alkylene, heteroalkylene, cycloalkylene, cycloheteroalkylene, arylene and heteroarylene, each of which is optionally substituted.

As used herein, "heteroalkylene" represents a group resulting from the substitution of one or more carbon atoms in a linear or branched alkylene group with an atom independently selected in each case from the group consisting of oxygen, nitrogen, phosphorus and sulfur.

[0040] In various embodiments described herein, a synthetic collagen-binding peptidoglycan can be administered to a patient (eg, a patient in need of treatment to inhibit platelet activation, such as that involved in thrombosis , exposed collagen binding platelets of the denoted endothelium, thrombosis, inflammation resulting from undressing the endothelium, intimal hyperplasia or vasospasm). In various embodiments, the synthetic collagen-binding peptidoglycan can be administered intravenously or in the muscle, for example. Suitable routes for parenteral administration include intravascular, intravenous, intraarterial, intramuscular, cutaneous, subcutaneous, percutaneous, intradermal and intraepidermal administration. Suitable means for parenteral administration include needle injectors (including microneedles), infusion techniques and catheter-based administration.

[0041] In an illustrative embodiment, pharmaceutical formulations for use with synthetic collagen-binding peptidoglycans for parenteral administration or catheter-based delivery comprising: a) a pharmaceutically active amount of the collagen-binding synthetic peptidoglycan; b) a pharmaceutically acceptable pH buffering agent to provide a pH in the range of about pH 4.5 to about pH 9; c) an ionic strength modifying agent in the concentration range of around Oto about 300 millimolar; and d) water soluble viscosity modifying agent in the concentration range of about 0.25% to about 10% of the total weight of the formula or any individual component a), b), c) or d) or any combination of a ), b), c) and d) are provided.

[0042] In various embodiments described herein, the pH buffering agents for use in the compositions and methods described herein are those agents known to those skilled in the art and include, for example, acetate, borate, carbonate. , citrate and phosphate buffers, as well as hydrochloric acid, sodium hydroxide, magnesium oxide, monopotassium phosphate, bicarbonate, ammonia, carbonic acid, hydrochloric acid, sodium citrate, citric acid, acetic acid, disodium hydrogen phosphate, borax, boric acid, sodium hydroxide, diethyl barbituric acid and proteins, as well as various biological buffers, for example, TAPS, Bicine, Tris, Tricine, HEPES, TES, MOPS, PIPES, cacodylate or MES.

[0043] In various embodiments described herein, ionic strength modifying agents include those agents known in the art, for example, glycerin, propylene glycol, mannitol, glucose, dextrose, sorbitol, sodium chloride, potassium chloride , and other electrolytes.

[0044] Useful viscosity modulating agents include ionic and non-ionic water soluble polymers; crosslinked acrylic acid polymers such as the family of "carbomer" polymers, for example, carboxypolyalkylenes which can be obtained commercially under the trademark Carbopol®; hydrophilic polymers such as polyethylene oxides, polyoxyethylene-polyoxypropylene copolymers and polyvinyl alcohol; cellulosic polymers and cellulosic polymer derivatives such as hydroxypropylcellulose, hydroxyethylcellulose, hydroxypropylmethylcellulose, hydroxypropylmethylcellulose phthalate, methylcellulose, carboxymethylcellulose and etherified cellulose; gums such as tragacanth and xanthan gum; sodium alginate; gelatin, hyaluronic acid and its salts, chitosans, gellan or any combination thereof.

[0045] Typically, non-acidic viscosity enhancing agents, such as a neutral or basic agent, are employed in order to facilitate the achievement of the desired pH of the formulation.

[0046] In various embodiments described herein, parenteral formulations may be suitably formulated as a sterile non-aqueous solution or as a dry form to be used in conjunction with a suitable vehicle such as sterile, pyrogen-free water. The preparation of parenteral formulations under sterile conditions, for example, by lyophilization, can be easily accomplished using standard pharmaceutical techniques well known to those skilled in the art.

[0047] In various embodiments described herein, the solubility of a synthetic collagen-binding peptidoglycan used in the preparation of a parenteral formulation can be increased by the use of appropriate formulation techniques, such as the incorporation of compositions that improve solubility. , such as mannitol, ethanol, glycerin, polyethylene glycols, propylene glycol, poloxomers and others known to those skilled in the art.

[0048] In various embodiments described herein, formulations for parenteral administration may be formulated to be immediate and / or modified release. Modified release formulations include delayed, sustained, pulsed, controlled, directed and programmed release formulations. Therefore, a synthetic peptidoglycan that binds to collagen can be formulated as a solid, semi-solid or thixotropic liquid for administration as an implanted reservoir that provides a modified release of the active compound. Illustrative examples of such formulations include drug-coated stents and microspheres of copolymer (dl-lactic, glycolic) acid (PGLA). In another embodiment, synthetic collagen-binding peptidoglycans or Compositions comprising synthetic collagen-binding peptidoglycan can be administered continuously, when appropriate.

[0049] In any of the embodiments described herein, synthetic collagen-binding peptidoglycan can be administered intravascularly in the patient (eg, in an artery or vein) in any suitable manner. In various embodiments described herein, the synthetic collagen-binding peptidoglycan can be administered in a patient's vessel before, during or after vascular intervention. In various embodiments, vascular interventions, such as percutaneous coronary intervention (PCI), may include, for example, stent placement, atherectomy, grafting and angioplasty, such as balloon angioplasty. Illustratively, the vascular intervention may be one that involves temporarily occluding an artery, such as a coronary artery or a vein (for example, balloon angioplasty), or one that does not involve temporarily occluding an artery or a vein (for example, angioplasty procedures not with balloon, stent placement procedures that do not involve balloon angioplasty, etc.). Illustrative modes of administration may include a catheter, parenteral administration, a coating on a balloon, through a porous balloon, a coated stent, and any combination thereof or any other known method of administering medications during an intervention procedure. vascular. In an illustrative embodiment, the target vessel may include a coronary artery, for example, any blood vessel that supplies blood to a patient's heart tissue, including native coronary arteries, as well as those that have been grafted into the patient, for example, in a previous coronary artery bypass procedure.

[0050] In any of the embodiments described herein, the target vessel in which the synthetic collagen-binding peptidoglycan is to be administered and where the vascular intervention procedure is to be performed may contain a blockage, such as a stricture or some other form of complete or partial blockage that causes reduced blood flow through the vessel. Thus, the synthetic peptidoglycan that binds to the collagen can be administered to the vessel through a catheter (for example, a dilatation catheter, an angioplasty balloon catheter on the wire, an infusion catheter, an exchange catheter fast or monorail, or any other catheter device known in the art) that is inserted percutaneously into the patient and that is screwed through the patient's blood vessels to the target vessel. Various catheter-based devices are known in the art, including those described in US Pat. No. 7,300,454. In several embodiments described herein where a catheter is used, the catheter used to administer the synthetic peptidoglycan that binds to the collagen may be the same catheter through which the vascular intervention will be performed, or it may be a different catheter (by example, a different catheter) that is inserted percutaneously into the patient through the same or a different skin incision and / or that is screwed through the patient's blood vessels to the target vessel through the same or a route different). In another embodiment, the synthetic collagen-binding peptidoglycan can be injected directly into the target vessel. In another embodiment, the synthetic collagen-binding peptidoglycan can be administered systemically (i.e., not administered directly to the target vessel, but administered by parenteral administration without catheter-based administration).

[0051] In the case where the container contains an obstruction (for example, a stenosis), administration can be carried out by delivering the synthetic collagen-binding peptidoglycan directly to the target vessel at the site of the obstruction or distal to the obstruction or both. In another embodiment, the synthetic collagen-binding peptidoglycan can be administered to one or more sites near the blockage. Illustratively, the tip of the catheter can be held stationary while administering the synthetic collagen-binding peptidoglycan, or the tip of the catheter can be moved while the synthetic collagen-binding peptidoglycan is being administered (e.g., in a proximal direction from a position that is initially distal to the block, to or through the block, or to a position that is proximal to the block).

[0052] As indicated above, in one embodiment, the synthetic collagen-binding peptidoglycan can be administered directly into the patient's vessel at a time before vascular intervention, for example, percutaneous coronary intervention. For example, administration of the synthetic collagen-binding peptidoglycan can be performed just before the vascular intervention (for example, within about 1 hour, as within approximately 30 minutes, within approximately 15 minutes and / or within

about 5 minutes before the vascular intervention). Optionally, the delivery of the synthetic collagen-binding peptidoglycan directly to the target vessel can be continued during all or part of the vascular intervention procedure and / or after the completion of said procedure, or the supply of the synthetic collagen-binding peptidoglycan directly to the Target Valuation can be stopped before the start of the vascular intervention procedure and not restarted later. In any of the embodiments described herein, the delivery of the synthetic collagen-binding peptidoglycan can be continuous or can be accomplished through a single or multiple administrations. Before, during and / or after the synthetic collagen binding peptidoglycan is administered to the target vessel, the same collagen binding synthetic peptidoglycan or one or more other synthetic collagen binding peptidoglycans can be administered.

[0053] In any of the embodiments described herein, the synthetic collagen binding peptidoglycan can be administered alone or in combination with suitable pharmaceutical carriers or diluents. The diluent or carrier ingredients used in the formulation of synthetic collagen-binding peptidoglycan can be selected so as not to diminish the desired effects of synthetic-binding peptidoglycan. collagen The collagen-binding synthetic peptidoglycan formulation may be in any suitable form. Examples of suitable dosage forms include aqueous solutions of the collagen-binding peptidoglycan, for example, a solution in isotonic saline, 5% glucose or other well-known pharmaceutically acceptable liquid carriers such as alcohols, glycols, esters and amides.

[0054] Suitable dosages of synthetic collagen-binding peptidoglycan can be determined by standard methods, for example by establishing dose-response curves in laboratory animal models or in clinical trials. Illustratively, suitable doses of synthetic collagen-binding peptidoglycan (administered in a single bolus or over time) include from 1 ng / kg to approximately 10 mg / kg, 100 ng / kg to approximately 1 mg / kg, approximately 1 pg / kg at about 500 pg / kg, or from about 100 pg / kg to about 400 pg / kg. In each of these embodiments, dose / kg refers to the dose per kilogram of mass or body weight of the patient. In other illustrative aspects, effective doses may range from about 0.01 pg to about 1000 mg per dose, 1 pg to about 100 mg per dose, or from about 100 pg to about 50 mg per dose, or from about 500 pg to about 10 mg per dose or about 1 mg to 10 mg per dose, or about 1 to about 100 mg per dose, or about 1 mg to 5000 mg per dose, or about 1 mg to 3000 mg per dose, or from about 100 mg to 3000 mg per dose, or from about 1000 mg to 3000 mg per dose.

[0055] Vascular intervention, such as percutaneous coronary intervention, can be carried out by any conventional procedure before, during, or after administration of the collagen-binding synthetic peptidoglycan. Examples of vascular intervention procedures contemplated for use in conjunction with the method of the present invention include stent placement, atherectomy and angioplasty, such as balloon angioplasty. The vascular intervention procedure may be one that involves temporary occlusion of the vessel (e.g., balloon angioplasty), or one that does not involve temporary occlusion of the vessel (e.g., balloonless angioplasty procedures, placement of stents that do not involve balloon angioplasty, etc.). Illustrative modes of administration may include a catheter, parenteral administration, a coating on a balloon, through a porous balloon, a coated stent, and any combination thereof or any other known method of administering medications during an intervention procedure. vascular.

[0056] In any of the embodiments described herein, kits for performing vascular intervention, such as the kits described above, are contemplated. The kits may include a catheter or stent and a synthetic peptidoglycan that binds to collagen. The synthetic collagen-binding peptidoglycan can be provided in any of the formulations discussed above and in the amount necessary to carry out our unique vascular intervention, such as from 1 ng / kg to approximately 10 mg / kg, from 100 ng / kg to approximately 1 mg / kg, from approximately 1 pg / kg to approximately 500 pg / kg, or from approximately 100 pg / kg to approximately 400 pg / kg. In each of these embodiments, dose / kg refers to the dose per kilogram of mass or body weight of the patient. In various embodiments described herein, the effective doses provided in the formulations may range from about 0.01 pg to about 1000 mg per dose, 1 pg to about 100 mg per dose, or from about 100 pg to about 50 mg per dose, or about 500 pg to about 10 mg per dose or about 1 mg to 10 mg per dose, or about 1 to about 100 mg per dose, or about 1 mg to 5000 mg per dose, or about 1 mg to 3000 mg per dose, or about 100 mg to 3000 mg per dose, or about 1000 mg to 3000 mg per dose. Manufacturing articles are also contemplated for any of these embodiments.

[0057] In any of the embodiments of kit or articles of manufacture described herein, the kit or article of manufacture may comprise a dose or multiple doses of collagen-binding synthetic peptidoglycan. The synthetic peptidoglycan that binds to the collagen can be in a primary container, for example, a glass vial, such as an amber glass vial with a rubber stopper and / or a removable aluminum seal. In another embodiment, the primary container may be plastic or aluminum, and the primary container may be sealed. In another embodiment, the primary container may be contained within a secondary container to further protect the composition from light.

[0058] In any of the embodiments described herein, the kit or article of manufacture that may contain instructions for use. Other suitable components of the kit or article of manufacture include excipients, disintegrants, binders, salts, local anesthetics ( e.g., lidocaine), diluents, preservatives, chelating agents, buffers, tonicity agents, antiseptic agents, wetting agents, emulsifiers, dispersants, stabilizers. These components may be available separately or mixed with the synthetic collagen-binding peptidoglycan. Any of the compositional embodiments described herein can be used to formulate the kit or article of manufacture.

[0059] In various embodiments described herein, the kit may contain more than one catheter or stent and a plurality of separate containers, each containing sterilizing synthetic collagen-binding peptidoglycan formulations in an amount necessary to carry out one or more vascular interventions Any type of stent or catheter can be included with the kit, including, for example, dilatation catheters, angioplasty balloon catheters on the wire, infusion catheters, rapid exchange catheters or monorail.

[0060] It is also contemplated that any of the formulations described herein may be used to administer the synthetic collagen-binding peptidoglycan (for example, one or more types) or in the absence or presence of a catheter-based device. The synthetic proteoglycan that binds to collagen can be formulated in an excipient. In any of the embodiments described herein, the excipient may have a concentration ranging from about 0.4 mg / ml to about 6 mg / ml. In various embodiments, the concentration of the excipient may range from about 0.5 mg / ml to about 10 mg / ml, about 0.1 mg / ml to about 6 mg / ml, about 0.5 mg / ml to about 3 mg / ml, approximately 1 mg / ml to approximately 3 mg / ml, approximately 0.01 mg / ml to approximately 10 mg / ml and approximately 2 mg / ml to approximately 4 mg / ml.

[0061] In various embodiments described herein, the dose of synthetic collagen-binding peptidoglycan may vary significantly depending on the condition of the patient, the disease condition to be treated, the route of administration and distribution in the tissues, and the possibility of co-use of other therapeutic treatments. The effective amount to be administered to a patient is based on the area of the body surface, the weight or mass of the patient and the medical evaluation of the patient's condition. In several exemplary embodiments, an effective dose may range from about 1 ng / kg to about 10 mg / kg, 100 ng / kg to about 1 mg / kg, from about 1 pg / kg to about 500 pg / kg, or from about 100 pg / kg up to approximately 400 pg / kg. In each of these embodiments, dose / kg refers to the dose per kilogram of body mass or body weight of the patient. In other illustrative aspects, effective doses may vary from about 0.01 pg to about 1000 mg per dose, 1 pg to about 100 mg per dose, or

about 100 pg to about 50 mg per dose, or about 500 pg to about 10 mg per dose or about 1 mg to 10 mg per dose, or about 1 to about 100 mg per dose, or about 1 mg to 5000 mg per dose, or about 1 mg to 3000 mg per dose, or about 100 mg to 3000 mg per dose, or about 1000 mg to 3000 mg per dose. In any of the various embodiments described herein, effective doses may range from about 0.01 pg to about 1000 mg per dose, 1 pg to about 100 mg per dose, about 100 pg to about 1.0 mg, about 50 pg to about 600 pg, about 50 pg to about 700 pg, about 100 pg to about 200 pg, about 100 pg to about 600 pg, about 100 pg to about 500 pg, about 200 pg to about 600 pg, or about 100 pg at about 50 mg per dose, or about 500 pg at about 10 mg per dose or about 1 mg to 10 mg per dose. In other illustrative embodiments, the effective doses may be 1 pg, 10 pg, 25 pg, 50 pg, 75 pg, 100 pg, 125 pg, 150 pg, 200 pg, 250 pg, 275 pg, 300 pg, 350 pg, 400 pg, 450 pg, 500 pg, 550 pg, 575 pg, 600 pg, 625 pg, 650 pg, 675 pg, 700 pg, 800 pg, 900 pg, 1.0 mg, 1.5 mg, 2.0 mg, 10 mg, 100 mg, or 100 mg at 30 grams.

[0062] Any effective regimen for the administration of synthetic collagen-binding peptidoglycan can be used. For example, synthetic collagen-binding peptidoglycan can be administered as a single dose or as a daily multiple dose regimen. In addition, a staggered regimen may be used, for example, one to five days per week as an alternative to daily treatment.

[0063] In various embodiments described herein, the patient is treated with multiple injections of the synthetic collagen-binding peptidoglycan. In one embodiment, the patient is injected several times (for example, about 2 to about 50 times) with the synthetic collagen-binding peptidoglycan, for example, at intervals of 12-72 hours or at intervals of 48-72 hours. Additional injections of synthetic peptidoglycan that binds to the collagen may be administered to the patient at an interval of days or months after the initial injections.

[0064] In any of the embodiments described herein, it is understood that a combination of two or more synthetic collagen-binding peptidoglycans, which differ in the peptide part, the glycan portion, or both, may be used instead of a single synthetic collagen-binding peptidoglycan.

[0065] It will also be appreciated that in the above embodiments, certain aspects of the compounds, compositions and methods are presented in the alternative in lists, such as, illustratively, the selections for any one or more of G and P. Therefore It should be understood that several alternative embodiments of the invention include individual members of those lists, as well as the various subsets of those lists. Each of these combinations should be understood as described in this document through the lists.

[0066] In the following illustrative examples, the terms "synthetic peptidoglycan" and "conjugate" are used synonymously with the term "synthetic collagen-binding peptidoglycan."

EXAMPLE 1

Peptide synthesis

[0067] All peptides were synthesized using a Symphony peptide synthesizer (Protein Technologies, Tucson, AZ), using an Fmoc protocol on a Knorr resin. The crude peptide was released from the resin with TFA and purified by reverse phase chromatography on an AKTAexplorer (GE Healthcare, Piscataway, NJ) using a Grace-Vydac 218TP C-18 reverse phase column and a water / acetonitrile gradient at 0.1% TFA. Dansyl modified peptides were prepared by adding an additional coupling step with dansyl-Gly (Sigma) before resin release. Peptide structures were confirmed by mass spectrometry. The following peptides were prepared as described above: RRANAALKAGELYKSILYGC, SYIRIADTNIT, Dansilo-GRRANAALKAGELYKSILYGC, and Dansilo-GSYIRIADTNIT. These peptides are abbreviated SILY, SYIR, Z-SILY and Z-SYIR. A biotin-labeled Z-SYIR peptide has also been synthesized using protocols known in the art and the peptide is terminated in amide. Additional peptides, KELNLVYTGC (abbreviated KELN) and GSITTIDVPWNVGC (abbreviated GSIT) were prepared as described above or purchased (GenScript, Piscataway, NJ). EXAMPLE 2

Conjugation of SILY to dermatan sulfate

PDPH attachment to oxDS

[0068] Bifunctional crosslinking CPPD (Pierce), reactive against sulfhydryl and amine groups, was used to conjugate SILY to oxDS. In the first step of the reaction, oxDS was dissolved in a coupling buffer (0.1 M sodium phosphate, 0.25 M sodium chloride, pH 7.2) to a final concentration of 1.2 mM. PDPH was added in a 10-fold molar excess, and the reaction proceeded at room temperature for 2 hours. The excess PDPH (MW 229 Da) was separated by gel filtration in an Akta Purifier using an XK 26-40 column packed with Sephadex G-25 medium and equilibrated with MilliQ water. The eluent was monitored at 215 nm, 254 nm and 280 nm. The first elution peak containing DS-PDPH was collected and lyophilized to conjugate it with SILY.

Conjugation of SILY

[0069] The peptide was dissolved in a 5: 1 molar excess in coupling buffer at a final peptide concentration of approximately 1 mM (limited by the solubility of the peptide). The reaction was allowed to proceed at room temperature overnight, and the excess peptide was separated and the DS-SILY conjugate was isolated by gel filtration as described above. See Figure 14 which shows a SILY / DS ratio of 1.06 after coupling.

EXAMPLE 3

Conjugation of Z-SILY to dermatan sulfate

[0070] Dermatan sulfate was conjugated with Z-SILY according to the method of Example 2.

REFERENCE EXAMPLE 4

Conjugation of keln to dermatan sulfate

[0071] Dermatan sulfate was conjugated to Keln according to the method of Example 2.

REFERENCE EXAMPLE 5

Conjugation of GSIT to dermatan sulfate

[0072] Dermatan sulfate was conjugated to GSIT according to the method of Example 2.

REFERENCE EXAMPLE 6

Conjugation of Z-SYIR to dermatan sulfate

[0073] Dermatan sulfate was conjugated to Z-SYIR using a method similar to that described in EXAMPLE 2.

EXAMPLE 7

Conjugation of SILY to heparin

[0074] Oxidized heparin (oxHep) (MW = 19.7kDa) containing 1 aldehyde per molecule (purchased from Celsus Laboratories, Cincinnati, OH). Additional aldehydes were formed by further oxidation in sodium meta-periodate as follows. oxHep was dissolved in 0.1 M sodium acetate, pH 5.5, at a concentration of 10 mg / ml. Then sodium metastate was added at a concentration of 2 mg / ml and allowed to react for 4 hours at temperature environment protected from light. Excess sodium meta-periodate was removed by desalination using a HiTrap size exclusion column (GEHealthcare) and the oxHep was lyophilized protected from light until conjugation with PDPH.

[0075] The oxHep was conjugated to PDPH by the method described for the DS-PDPH conjugation, EXAMPLE 2. The PDPH was reacted in a 50-fold molar excess. To achieve a higher concentration of PDPH, 10 mg of PDPH was dissolved in 75 pL of DMSO and mixed with 1 ml of coupling buffer containing oxHep. The reaction proceeded at room temperature for 2.5 hours and the excess PDPH was removed by desalination. Heparin containing PDPH (Hep-PDPH) was stored as a lyophilized powder until it reacted with SILY.

[0076] SILY was reacted in a 10-fold molar excess with Hep-PDPH as described for DS-SILY conjugation in EXAMPLE 2. The reaction was monitored as described for DS-SILY in EXAMPLE 2 and showed 5 44 conjugated SILY peptides per heparin molecule as shown in Figure 13.

REFERENCE EXAMPLE 8

Conjugation of GSIT with heparin

[0077] Heparin was conjugated to GSIT according to the method of EXAMPLE 7 (abbreviated Hep-GSIT).

EXAMPLE 9

Conjugation of SILY to dextran

[0078] Dextran was conjugated to SILY according to the method of EXAMPLE 7 that replaces heparin with dextran. Modification of the conditions for the oxidation of dextran with sodium meta-periodate in the first step to allow the preparation of conjugates with different molar ratios of SILY to dextran. For example, dextran-SILY conjugates with a molar ratio of SILY to dextran of about 6 and a dextran-SILY conjugate with a molar ratio of SILY to dextran of about 9 (abbreviated Dex-SILY6 and Dex-SILY9) were prepared.

EXAMPLE 10

Conjugation of SILY to hyaluronano

[0079] The hyaluronan was conjugated to SILY according to the method of Example 7 (abbreviated HA-SILY).

EXAMPLE 11

SILY to Collagen (Biacore)

[0080] Biacore studies were performed on a Biacore 2000 using a CM-3 chip (Biacore, Inc., Piscataway, NJ). The CM-3 chip is coated with covalently bonded carboxymethylated dextran, which allows the binding of the collagen substrate through free amine groups. Flow cells (FCs) 1 and 2 were used, with FC-1 as the reference cell and FC-2 as a cell immobilized with collagen. Each FC was activated with EDC-NHS, and 1500 RU of collagen was immobilized on FC-2 by flowing 1 mg / ml of collagen in sodium acetate, pH 4, buffer at 5 pL / min for 10 min. The unreacted NHS ester sites were covered with ethanolamine; The FC-1 control was activated and covered with ethanolamine.

[0081] To determine peptide binding affinity, SILY was dissolved in 1x HBS-EP buffer (Biacore) in different concentrations from 100 pm to 1.5 pm in 2-fold dilutions. The flow rate was maintained at 90 pL / min, which is in the range suggested by Myska to determine the binding kinetics (Myska, 1997). The first 10 injections were buffer injections, which help to prime the system, followed by random sample injections, which are performed in triplicate. The analysis was performed with the BIAevaluation software (Biacore). Representative association / dissociation curves are shown in Figure 3, demonstrating that the SILY peptide reversibly binds with collagen. K ^d = 1.2 pm was calculated from the on-off junction kinetics.

EXAMPLE 12

Z-SILY binding to collagen

[0082] Binding assays were performed on a black 96 well high junction plate with a transparent bottom (Costar). Collagen was compared with untreated wells and wells coated with BSA. Collagen and BSA were immobilized at 37 ° C for 1 hour by incubating 90 pl / well at concentrations of 2 mg / ml in 10 mM HCl and 1xPBS, respectively. Each well was washed 3x with 1xPBS after incubation. Z-SILY was dissolved in 1 x PBS at concentrations of 100 | jm at 10 nM in 10-fold dilutions. The wells were incubated for 30 minutes at 37 ° C and 3X rinsed with PBS and then filled with 90 ml of IxPBS. Fluorescence readings were taken on a Spectramax M5 spectrophotometer (Molecular Devices) at excitation / emission wavelengths of 335 nm / 490 nm, respectively. The results are shown in Figures 4 and 5. Kd = 0.86 jm was calculated from equilibrium kinetics.

EXAMPLE 13

Characterize DS-SILY

[0083] To determine the number of SILY molecules conjugated to DS, the production of pyridine-2-thione was measured using a modified protocol provided by Pierce. Dermatan sulfate was dissolved with 1.1 PDPH molecules bound in coupling buffer (0.1 M sodium phosphate, 0.25 M sodium chloride) at a concentration of 0.44 mg / ml and the absorbance was measured at 343 nm using a SpectraMax M5 (Molecular Devices). SILY was reacted in a molar excess of 5 times and the absorbance measurements were repeated immediately after the addition of SILY and after allowing to react for 2 hours. To ensure that SILY is not absorbed at 343 nm, the coupling buffer containing 0.15 mg / ml SILY was measured and compared with the absorbance of the buffer alone.

[0084] The number of SILY molecules conjugated to DS was calculated the extinction coefficient of pyridine-2-thione using the following equation (Abs343 / 8080) X (MWDs / DSmg / ml). The results are shown in FIGURE 14.

EXAMPLE 14

Collagen binding, fluorescence data - DS-SILY

[0085] In order to determine whether the peptide conjugate maintained its ability to bind collagen after its conjugation with DS, a fluorescent binding assay was performed. A fluorescently labeled version of SILY, Z-SILY, was synthesized, adding dansylglycine to the term amine. This peptide was conjugated to DS and purified using the same methods described for SILY.

[0086] Binding assays were performed on a black 96 well high junction plate with a transparent bottom (Costar). Collagen was compared with untreated wells and wells coated with BSA. The monomeric collagen (Advanced Biomatrix Cat. No. 5010) and BSA were immobilized at 37 ° C for 1 h by incubating 90 jL / well at concentrations of 2 mg / ml in 10 mM HCl and 1xPBS respectively. Each well was washed 3x with 1xPBS after incubation.

[0087] The wells were pre-incubated with DS at 37 ° C for 30 minutes to eliminate non-specific binding of DS to collagen. The wells were rinsed 3x with 1xPBS before incubation with DS-Z-SILY. DS-Z-SILY was dissolved in 1 x PBS at concentrations of 100 jM to 10 nM in 10-fold dilutions. The wells were incubated for 30 min at 37 ° C and rinsed 3x and then filled with 90 µl of 1xPBS. Fluorescence readings were taken on a Spectramax M5 spectrophotometer (Molecular Devices) at excitation / emission wavelengths of 335 nm / 490 nm, respectively.

[0088] The fluorescence binding of DS-Z-SILY in immobilized collagen, BSA, and untreated wells are compared in FIGURE 7. The results show that DS-Z-SILY specifically binds to wells treated with collagen against BSA and untreated wells. The untreated wells of the high binding plate were designed to be a positive control, although little binding was observed in relation to the wells treated with collagen. These results suggest that SILY maintains its ability to bind to collagen after its conjugation with DS. Preincubation with DS did not prevent binding, suggesting that the conjugate binds separately to the DS alone.

EXAMPLE 15

Preparation of type I collagen gels

[0089] The gels were prepared with Nutragen collagen (Inamed, Freemont, CA) at a final concentration of 4 mg / ml collagen. The Nutragen pool is 6.4 mg / mL in 10 mM HCl. The gel preparation was performed on ice and fresh samples were made before each test. The collagen solution was adjusted to physiological pH and salt concentration, adding appropriate volumes of 10x PBS (phosphate buffered saline), 1xPBS and 1M NaOH. For most of the experiments, samples of DS, decorin, DS-SILY or DS-SYIR were added to a 10: 1 collagen sample molar ratio by a final addition of 1xPBS (equal volumes throughout the treatments) in which test samples dissolved at appropriate concentrations. In this way, the samples are constantly maintained at pH 7.4 and the physiological salt concentration. Collagen samples only received an addition of 1xPBS without any dissolved sample. Fibrinogenesis will be induced by incubating neutralized collagen solutions at 37 ° C overnight in a humidified chamber to avoid dehydration. Collagen gel solutions: sample molar ratios other than 10: 1 were prepared similarly.

EXAMPLE 16

Viscoelastic characterization of type III collagen containing gels

[0090] Gels containing type III collagen were prepared as in EXAMPLE 15 with the following modifications: treated and untreated gel solutions were prepared using a collagen concentration of 1.5 mg / ml (90% type III collagen) (Millipore), 10% type I collagen), 200 pl samples were pipetted into wettable surfaces of 20 mm diameter of slides printed with hydrophobic material. These solutions were allowed to gel at 37 ° C for 24 hours. The gels were formed from collagen alone, collagen sulfate treated collagen (1: 1 and 5: 1 molar ratio), and collagen treated with collagen III binding peptides alone (GSIT and KELN, 5: 1 molar ratio) They served as controls. The treated gels contained the peptidoglycans (DS-GSIT or DS-KELN at 1: 1 and 5: 1 molar ratios. All proportions are collagen proportions: treatment compound. The gels were characterized as in EXAMPLE 18, except that the samples were analyzed along the frequency range of 0.1 Hz to 1.0 Hz with a controlled stress of 1.0 Pa. As shown in Figures 8 and 9, the dermatan-GSIT sulfate conjugate and the conjugate Dermatan-KELN sulfate (synthetic peptidoglycans) can influence the viscoelastic properties of gels formed with type III collagen.

EXAMPLE 17

Fibrinogenesis

[0091] Collagen fibrinogenesis was monitored by measuring the turbidity-related absorbance at 313 nm, providing information on the fibrilogenesis rate and the diameter of the fibril. Gel solutions were prepared as described in EXAMPLE 15 (4 mg / ml collagen, 10: 1 collagen: treatment, unless otherwise indicated) and 50 uL / well at 4 ° C was added to a plate of 384 wells. The plate was kept at 4 ° C for 4 hours before starting the formation of fibrils. A SpectraMax M5 at 37 ° C was used to measure the absorbance at 313 nm at 30 s intervals for 6 hours. The results are shown in Figure 10. Dermatan-SILY sulfate decreases the rate of fibrinogenesis.

EXAMPLE 18

Crio-SEM Measurements in Type III Collagen

[0092] Cryo-SEM gels were formed, as in Example 15, directly at the SEM stage and incubated at 37 ° overnight with the following modifications. The collagen concentration was 1 mg / ml (90% of type III collagen, 10% of type I collagen). The collagen: DS ratio was 1: 1 and the collagen: peptidoglycan ratio was 1: 1. The images were recorded as in EXAMPLE 19. The relationship between the volume of vacuum and the volume of fibrils was measured using a variation of the method of EXAMPLE 28. The results are shown in Figures 11 and 12. Dermatan sulfate-KELN and Dermatan-GSIT sulfate decrease the vacuum space (increase the diameter of the fibril and branching) in the treated collagen gels.

EXAMPLE 19

AFM D-band confirmation

[0093] Gel solutions were prepared as described in Example 15 and 20 pL of each sample were pipetted onto a glass slide and allowed to gel overnight in a humidified incubator. The gels were dehydrated by treatment with graduated ethanol solutions (35%, 70%, 85%, 95%, 100%), 10 min in each solution. AFM images were made in contact mode, with a scan rate of 2 Hz (SPM multimode, Veeco Instruments, Santa Barbara, CA, USA, AFM tips, silicon nitride contact mode tip k = 0.05N / m, Veeco Instruments) Deviation nominal value: 0-1 volts. Band D was confirmed in all treatments as shown in Figures 2 and 26.

EXAMPLE 20

Collagen remodeling

Preparation of tissue samples

[0094] Following a method by Grassl, et al. (Grassl, et al., Journal of Biomedical Materials Research 2002, 60, (4), 607-612), collagen gels were formed with or without synthetic PG mimics as described in EXAMPLE 15. Human aortic smooth muscle cells (Cascade Biologies, Portland, OR) were seeded into collagen gels by adding 4 x 106 cells / ml to the neutralized collagen solution before incubation. The cell collagen solutions were pipetted into an 8-well Lab-Tek slide and incubated in a humidified incubator at 37 ° C and 5% CO2. After gelation, the cell collagen gels will be covered with 1 ml of Medium 231 as prescribed by Cascade. Every 3-4 days, the medium was removed from the samples and the hydroxyproline content was measured by a standard hydroxyproline assay (Reddy, 1996).

Hydroxyproline content

[0095] To measure the degraded collagen in the supernatant medium, the sample was lyophilized, the sample hydrolyzed in 2M NaOH at 120 ° C for 20 min. After cooling, the free hydroxyproline was oxidized by adding chloramine-T (Sigma) and reacting for 25 minutes at room temperature. The Ehrlich aldehyde reagent (Sigma) was added and allowed to react for 20 minutes at 65 ° C, and then the absorbance at 550 nm was read on an M-5 spectrophotometer (Molecular Devices). The hydroxyproline content in the medium is an indirect measure of the remodeling potential of collagen and degraded tissue. Cultures were incubated for up to 30 days and three samples of each treatment were measured. The gels incubated without added cells were used as control. The free SILY and De13 peptides resulted in greater degradation of collagen compared to collagen alone, measured by the hydroxyproline content in cellular media, as shown in FIGURE 39.

Cell viability

[0096] Cell viability was determined using a live / dead violet viability / vitality kit (Molecular Probes. The kit contains calcein-violet stain (live cells) and aqua-fluorescent reactive dye (dead cells). Samples were washed with 1 x PBS and incubated with 300 µl of dye solution for 1 hour at room temperature.To remove unbound dye, samples were rinsed with 1 x PBS.The live and dead cells were counted after imaging a portion 2 -D with filters 400/452 and 367/526 in an Olympus FV 1000 confocal microscope with a 20x objective.The gels were scanned for representative regions and 3 sets of images were taken at equal distances on the gel for all samples .

REFERENCE EXAMPLE 21

DS-Dc13 Preparation

[0097] The sequence of the DE13 peptide is SYIRIADTNITGC and its fluorescently labeled form is Zs YIR iADTNITGC, where Z designates Dansylglycine. The conjugation with dermatan sulfate using the PDPH heterobifunctional crosslinker is performed as described for DS-SILY in EXAMPLE 2. As shown in Figure 15, the molar ratio of Dc13 to dermatan sulfate in the conjugate (DS-Dc13) It was about 1.

Example 22

Fluorescence binding assay for DS-ZSILY

[0098] The fluorescence assays described for DS-ZSILY binding were performed with the peptide sequence ZSYIRIADTNITGC (ZDc13). The results appear in Figure 16, showing that DS-ZDc13 specifically binds to the surface of the collagen in a dose-dependent manner, although saturation was not achieved at the highest rate tested.

REFERENCE EXAMPLE 23

Fibrillogenesis assay for DS-Dc13

[0099] A fibrinogenesis assay as described for DS-SILY, Example 17, performed with the conjugated DS-DC13. The results shown in Figure 17 indicate that DS-Dc13 delays fibrinogenesis and decreases overall absorbance in a dose-dependent manner. The free Dc13 peptide, on the other hand, has little effect on fibrinogenesis compared to collagen only in the high molar ratio of collagen: additive of 1: 1. EXAMPLE 24

Use of Crio-SEM to measure the diameters of fibrils.

[0100] Using a modification of EXAMPLE 18, the diameters of the fibrils were measured by 8crio-SEM. The fiber diameters of the cryo-SEM images taken at 20,000x were measured using ImageJ (NIH) software. At least 45 fibrils were measured for each treatment. The results are presented as means ± SE. Statistical analysis was performed using DesignExpert software (StatEase) with a = 0.05. The results are shown in Figure 18. Decorin and synthetic peptidoglycans significantly decrease the diameter of the fibrils on the collagen or collagen sulfate dermatan. Compared to collagen alone, the free De13 peptide does not affect the diameter of the fibril, while free SILY causes a decrease in the diameter of the fibril.

EXAMPLE 25

Cell culture and gel compaction

[0101] Smooth human coronary artery muscle cells (HCA SMC) (Cascade Biologics) were grown in growth medium (Medium 231 supplemented with smooth muscle growth factor). The cells of passage 3 were used for all experiments. Differentiation medium (Medium 231 supplemented with 1% FBS and 1x pen / streptococcus) was used for all experiments, unless otherwise indicated. This medium differs from the manufacturer's protocol in that it does not contain heparin.

[0102] Collagen gels were prepared with each additive as described with the exception that the addition of the 1x PBS sample was omitted to accommodate the addition of the cells in media. After incubating on ice for 30 minutes, HCA SMC was added in differentiation medium to the gel solutions to a final concentration of 1 x 10 6 cells / ml. The gels were formed in quadruplicate on plates treated with 48-well tissue-free culture (Costar) for 6 hours before adding 500 pL / well differentiation medium. The gels were released from the edges of the well after 24 hours. The medium was changed every 2-3 days and the images for compaction were taken at the same time points using a Gel Doc System (Bio-Rad). The cross-sectional area of the circular gels that correlates with the degree of compaction was determined using ImageJ (NIH) software. Gels that did not contain cells were used as a negative control and the cells in collagen gels without additives were used as a positive control. The results are shown in Figure 19. On day 10, all gels had been compacted to approximately 10% of the original gel area, and the differences between the additives were small. Gels treated with DS-Dc13 were mild, but significantly less compact than gels treated with decorin or collagen, but the compaction was statistically equivalent to that observed with gels treated with DS and SIL-SILY.

EXAMPLE 26

Elastin measurement

[0103] Collagen gels seeded with HCA SMC were prepared as described in Example 25. The differentiation medium was changed every three days and the gels were cultured for 10 days. Collagen gels that did not contain cells were used as control. The gels were rinsed in 1xPBS overnight to remove serum protein and the gels were analyzed for elastin content using Fastin elastin analysis according to the manufacturers protocol (Biocolor, County Atrim, United Kingdom). Briefly, the gels were solubilized in 0.25 M oxalic acid by incubating at 100 ° C for 1 h. The elastin was precipitated and the samples were centrifuged at 11,000 x g for 10 min. The solubilized collagen supernatant was removed and the elastin sediment was stained with Fastin dye reagent for 90 minutes at room temperature. The samples were centrifuged at 11,000 x g for 10 minutes and the unbound dye in the supernatant was removed. The dye from the elastin granules was released by the Fastin Dye dissociation reagent, and the 100 ml samples were transferred to a 96-well plate (Costar). The absorbance was measured at 513 nm and the elastin content was calculated from a standard a-elastin curve. The results of these tests are shown in Figure 20. Treatment with DS-SILY significantly increased elastin production in all samples. Treatment with DS and DS-Dc13 significantly decreased elastin production on untreated collagen. Control samples of collagen gels without cells showed no elastin production.

EXAMPLE 27

Effect of heparin or heparin-SlLY on platelet interaction

[0104] The collagen was immobilized on glass cover slides (18 mm) by incubating slides with 2 mg / ml collagen in 10 mM HCl for 1 hour at 37 ° C. Then, the slides were washed with 1x PBS and stored at 4 ° C in 1x PBS for 24 hours until further tests were performed. Untreated glass cover slides were used as a negative control. The slides were placed on an untreated plate with 48-well tissue culture (Costar) with the collagen surface facing up. Heparin or heparin-SlLY was dissolved in 1x PBS at a concentration of 100 pm and incubated at 100 pL / well for 30 min at 37 ° C. Unbound heparin or heparin-SlLY were aspirated and the surfaces were washed with 1 ml of 1x PBS. Collagen immobilized slides incubated with 1x PBS containing no additive were used as a positive control.

[0105] Total human blood was centrifuged at 800 x g for 15 min and 100 pL of platelet rich plasma is removed from the leukocyte layer and added to each well. After incubating for 1 hour at 37 ° C, platelet rich plasma was extracted from the wells and the wells were gently washed with 1x PBS to remove unbound cells. The slides were fixed with 5% glutaraldehyde for 1 hour at room temperature, rinsed and lyophilized before imaging. The slides were coated by gold spraying for 3 min and 200x images were taken in a JEOL 840 SEM. The results are shown in Figure 21. These images show that treatment with the heparin-SlLY conjugate affects platelet binding to collagen.

EXAMPLE 28

Cryo-SEM measurement of fibrillar density

[0106] Collagen gels were formed in the presence of each additive in a 10: 1 molar ratio, as described in EXAMPLE 15, directly at the SEM stage, processed, and images were formed as described. The 10,000x images were analyzed for fibrile density calculations. The images were converted to 8 bits in black and white, and the threshold values for each image were determined using ImageJ (NIH) software. The threshold was defined as the value where all visible fibrils are white, and all empty space is black. The ratio of white to black area was calculated using MatLab software. All measurements were taken in triplicate and the thresholds were determined by an observer blinded to the treatment. The images of the gels are shown in Figure 25 and the measured densities are shown in FIGURE 22.

EXAMPLE 29

Viscoelastic characterization of gels containing Dc13 or DS-Dc13

[0107] Collagen gels were prepared, as in EXAMPLE 15. Viscoelastic characterization was performed as described in Example 16 in gels formed with varying ratios of collagen for additive (treatment). Treatment with dermatan sulfate or dermatan-Dc13 conjugate increases the stiffness of the resulting collagen gel on the untreated collagen, as shown in FIGURE 23.

EXAMPLE 30

Cell Proliferation and Cytotoxicity Assay

[0108] HCA SMCs, prepared as in EXAMPLE 25, were seeded at 4.8 x 104 cells / ml in growth medium on a black / transparent 96-well tissue culture bottom plate (Costar) and were They allowed to adhere for 4 hours. The growth medium was aspirated and 600 µl of differentiation medium containing each additive was added to each well at a concentration equivalent to the concentration within the collagen gels (1.4 x 10-6 M). Cells were incubated for 48 hours and then cytotoxicity and proliferation were analyzed using the live-dead and CyQuant (Invitrogen) assays, respectively, according to the manufacturer's protocol. Cells were used in differentiation medium that did not contain additive as a control. The results are shown in Figure 24 which indicates that none of the treatments demonstrated significant cytotoxic effects.

EXAMPLE 31

Inhibition of platelet binding and platelet activation to type I collagen

Microplate Preparation

[0109] Type I fibrillar collagen (Chronolog, Havertown, PA) was diluted in isotonic glucose at a concentration of 20-100 pg / ml. 50 µl of collagen solution was added to each well of a 96-well high junction plate. The plate was incubated overnight at 4 ° C and then rinsed 3X with 1X PBS.

[0110] Peptidoglycan was diluted in PBS IX at solution concentrations of 25 pm to 50 pm and 50 pm was added to the collagen coated wells. GAG, peptide or PBS controls were also added to collagen coated wells as controls. The treatments were incubated at 37 ° C with shaking at 200 rpm for 30 min. Then, the wells were rinsed 3X with IX PBS, including a 20 minute rinse with 200 rpm of stirring to remove the unbound treatment molecule.

Preparation and activation of platelets

[0111] Human blood was drawn from healthy volunteers by venipuncture following the approved Purdue IRB protocol and with informed consent. The first 5 ml of blood were discarded, since they can be contaminated with collagen and other proteins, and then approximately 15 ml were collected in citrated glass vacutainers (BD Bioscience). The blood was centrifuged in the glass tube for 20 minutes at 200 x g at 20 ° C. The top layer of centrifuged blood, platelet rich plasma (PRP), was used for platelet experiments. PRP (50 uL / well) was added to the microplate and allowed to incubate for 1 hour at room temperature without stirring.

[0112] After 1 hour of incubation, the PRP was removed from each well and added to a microcentrifuge tube containing 5 pL ETP (107 mM EDTA, 12 mM theophylline, and 2.8 m E1 M prostaglandin) to inhibit even more platelet activation. These tubes were rotated at 4 ° C for 30 minutes at 1900 x g to settle the platelets. The supernatant (platelet serum) was collected for ELISA studies to assess the presence of PF-4 and Nap-2 platelet activation markers.

Platelet Adhesion

[0113] After the PRP was removed from the wells of the coated collagen / treatment plates, the wells were rinsed 3 times with 0.9% NaCl for 5 min each stirring at 200 rpm. Platelet adhesion was quantified colorometrically or visualized by fluorescence.

Colorimetric Assay

[0114] 140 pl of a sodium citrate / citric acid buffer (0.1 M, pH 5.4) containing 0.1% Triton X-100 and 1 mg / ml p-nitrophenyl phosphate were added to each well. The background absorbance was measured at 405 nm. The plate was then incubated for 40 minutes at room temperature with shaking at 200 rpm. Triton X-100 creates pores in cells, which allows p-nitrophenyl phosphate to interact with acid phosphatase in platelets to produce pnitrophenol. After 40 minutes of incubation, 100 µl of 2 M NaOH was added to each well. The change in pH stops the reaction by inactivating acid phosphatase and also transforms p-nitrophenol into an optically active compound. The absorbance at 405 nm was then read and correlated with the number of adhered platelets. The results are shown in Figure 29.

Fluorescence test

[0115] The adhered platelets were fixed by incubation with 4% paraformaldehyde for 10 minutes at room temperature. The platelets were permeabilized with 0.1% Triton X-100 for 5 min. Platelet actin was labeled by incubation with phalloidin-AlexaFluor 488 (Invitrogen) containing 1% BSA for 30 min. The wells were rinsed 3X with IX PBS, and images of adhered platelets were taken using a vertical fluorescence microscope using a DAPI filter.

[0116] See FIGURES 30 to 39 for results. Platelet aggregation on untreated collagen surfaces is indicated by blurred images that result from clustered platelets. Without being limited by theory, it is believed that the clustering of platelets in the z-direction (perpendicular to the plate surface) prevents the capture of images in a focal plane. On treated surfaces, reduced platelet aggregation produces fewer agglomerations (less platelets in the z-direction) and focused images can be captured on the plate surface. These images show that treatment with synthetic peptidoglycans reduces the adhesion of platelet cells to collagen.

Detection of platelet activation markers

[0117] The supernatant (platelet serum) obtained after granulation of the platelets was used to determine the activation factors released. Platelet factor 4 (PF-4) and p-thromboglobulin (Nap-2) are two proteins contained in the platelet alpha granules that are released after platelet activation. Sandwich ELISA were used to detect each protein. The components for both sandwich ELISAs were purchased from (R&D Systems) and the protocols provided were followed. Serum platelet samples were diluted 1: 10,000-1: 40,000 in 1% BSA in IX PBS, so the values fell within a linear range. The results shown in Figures 27 and 28 show that treatment with synthetic peptidoglycans decreases platelet activation by type I collagen.

EXAMPLE 32

Inhibition of platelet binding and platelet activation to Type III and Type I collagen

[0118] The method according to Example 31 was used with the following modification.

Microplate Preparation

[0119] Type I collagen (rat tail collagen, BD Biosciences) and type III collagen (Millipore) were combined on ice with NaOH, 1X PBS, and 10X PBS at physiological conditions. The total collagen concentration was 1 mg / ml with 70% type I collagen and 30% type III collagen. 30 ml of the collagen solution was pipetted into each well of a 96-well plate. The plate was incubated at 37 ° C in a humidified incubator for one hour, allowing a gel composed of fibrillar collagen to form in the wells. The wells were rinsed 3X with 1X PBS.

[0120] The peptidoglycan was diluted in 1X PBS in solution concentrations 25vpm and 50vpm was added to the collagen coated wells. GAG, peptide or PBS controls were also added to collagen coated wells as controls. The combinations of peptidoglycan or peptide were composed of 25 pm of each molecule in 1X PBS. The treatments were incubated at 37 ° C with shaking at 200 rpm for 30 min. The wells were then rinsed 3X with 1X PBS, including a 10 minute rinse with 200 rpm shaking to remove the unbound treatment molecule.

[0121] The results of the platelet activation inhibition measurements shown in Figure 39 demonstrate that synthetic peptidoglycans inhibit platelet cell activation by a mixture of type I and type III collagen.

[0122] The results shown in Figure 40 demonstrate that peptidoglycans inhibit binding to mixtures of type I and type III collagen platelet cells.

EXAMPLE 33

Synthesis of peptidoglycan

[0123] The peptides used to synthesize the peptidoglycans described in this Example and the following Examples were synthesized by GenScript (Piscataway, New Jersey). Peptidoglycan was synthesized as described with modifications. Dermatan sulfate (DS) was oxidized by oxidation with periodate, in which the degree of oxidation was controlled by varying amounts of sodium meta-periodate. After being oxidized at room temperature for 2 hours protected from light, the oxidized DS was desalted in 1xPBS pH 7.2 by ^{size exclusion} chromatography ^{using a column packed with Bio-gel P} -6 (BioRad). The BMPH heterobifunctional crosslinker (Thermo Fischer Scientific) was added to oxidized DS in a 30-fold molar excess to DS, and reacted for 2 hours at room temperature protected from light. The intermediate DS-BMPH was purified after excess BMPH by size exclusion as described with 1xPBS pH 7.2 as an operating buffer. The number of BMPH crosslinkers bound to DS was calculated by BMPH consumption determined from the 215 nm peak area of the BMPH excess peak. A standard BMPH curve was generated to calculate excess BMPH. The SILY free peptide was dissolved in water at a concentration of 2 mg / ml and added in a molar excess to the number of BMPHs bound and reacted for 2 hours at room temperature. The final DS-SILYn product was purified by size exclusion using a column packed with Sephadex G-25 medium (GE Lifesciences) with Millipore water as an operating buffer. The final product was frozen immediately, lyophilized and stored at -20 ° C until further testing.

[0124] A biotin-labeled version of peptidoglycan was synthesized by reacting 2 moles of SILYbiotine per mole of DS-BMPH for 1 hour, followed by the addition of unlabeled SILY to a 1 molar excess by BMPH attached. After adding unlabeled SILY, the reaction continued for 2 hours at room temperature before purification. Biotin-labeled peptidoglycan is designated as DS-SILYn-biotin where n is the total number of SILY peptides per molecule. For DS-SILY4-biotin, only SILY marked with SILY was reacted, instead of unmarked biotin.

EXAMPLE 34

Purification and characterization of DS-BMPH.

[0125] Oxidized DS was coupled to BMPH as described and purified from excess BMPH by size exclusion chromatography. As shown in Figure 41, the amount of excess BMPH is calculated by integrating the excess BMPH peak and comparing with a standard curve for BMPH. As shown in Figure 42, as the amount of sodium meta-periodate varies, the number of BMPH crosslinkers per DS chain increases linearly.

EXAMPLE 35

Affinity of binding of peptidoglycan to collagen

[0126] Fibrillar collagen (Chronolog, Havertown, PA) was coated on the surface of a 96-well junction plate (Costar) at a concentration of 50 pg / ml diluted in isotonic glucose. The plates were covered and incubated overnight at 4 ° C. Unbound collagen was removed by rinsing 3 times with 1xPBS pH 7.4. The plates were blocked with 1% BSA for 3 hours at room temperature. The peptidoglycan was dissolved at varying concentrations in 1xPBS pH 7.4 containing 1% BSA and immediately added to the collagen surfaces, and allowed to incubate for 15 minutes at room temperature. The plates were rinsed 3 times with 1xPBS pH 7.4 containing 1% BSA. Streptavidin-HRP solution (R&D Systems, Minneapolis, MN) was then added to the plates and incubated for 20 minutes at room temperature. Unbound streptavidin was rinsed 3 times with 1xPBS pH 7.4 and 100 ml / well of color solution (stabilized hydrogen peroxide and stabilized tetramethylbenzidine, R&D Systems, Minneapolis, MN) were added to each well and incubated for 20 minutes at room temperature protected from light. The color evolution reaction was stopped with 50 pL of 2N sulfuric acid and the absorbance at 450 nm was measured using a Vis M5 UV spectrophotometer (Molecular Devices). The imperfections of the plates (540 nm) were subtracted from the absorbance values.

[0127] The binding affinities of biotin-labeled peptidoglycans, labeled using protocols known in the art, DS-SILY4 and DS-SILY18 were calculated by adjusting the saturation of the binding curves and calculating the inflection point. As shown in Figure 43, DS-SILY4 and DS-SILY18 bind to fibril collagen with a Kd of 118 nM and 24 nM, respectively. By increasing the number of peptides per DS skeleton, it is also evident that there are more molecules capable of binding to the surface of the collagen, which is observed by the increased absorbance of DS-SILY18 that contains no more biotin markers than DS-SILY4. Therefore, DS-SILY18 is expected to show a better inhibition of platelets, since it can form a denser cover of the collagen surface.

EXAMPLE 36

Inhibition of platelet binding and activation

[0128] Chronolog type I fibrillar collagen was diluted in isotonic glucose at a concentration of 50 pg / ml. 50 ml of collagen solution was added to each well of a high-well 96 well plate. The plate was incubated overnight at 4 ° C and then rinsed 3X with 1X PBS. For the microplate assays, the peptidoglycan was diluted in 1X PBS at concentrations between 0.0001 pm and 100 pm and a 50 pL solution was added to the collagen coated wells. DS, peptide or 1X PBS were also added to collagen coated wells as controls. The treatments were incubated at 37 ° C with shaking at 200 rpm for 15 min. Then, the wells were rinsed from the unbound treatment by removing the treatment solution, adding PBS and stirring the wells for 24 hours. During the 24 hours, the PBS solution was changed 3 times.

[0129] Human whole blood was collected from healthy volunteers by venous puncture. The first 5 ml of blood was discarded and then approximately 20 ml was collected in citrated glass vacutainers (BD Bioscience). The blood was centrifuged in the glass tube for 20 minutes at 200 g at 25 ° C. The top layer of centrifuged blood, platelet rich plasma (PRP), was used for platelet experiments.

[0130] PRP (50 uL / well) was added to the microplate for 1 hour at room temperature without stirring. After 1 hour of incubation, 45 pl of PRP were removed from each well and added to a microcentrifuge tube containing 5 ml of ETP (107 mM EDTA, 12 mM theophylline and E12.8 mM prostaglandin) to inhibit platelet activation additional. These tubes were rotated at 4 ° C for 30 minutes at 2000 g to settle the platelets. The supernatant (platelet serum) was collected for ELISA studies to assess the presence of PF-4 and NAP2 platelet activation markers. Sandwich ELISA were used to detect each protein. The components for both sandwich ELISAs were purchased from R&D Systems and the protocols provided were followed. The platelet serum was diluted 10,000 times in 1% BSA in 1X PBS so that the values were within a linear range.

[0131] Platelet activation was measured by the release of platelet factor 4 (PF4) and p-thromboglobulin (NAP2). Figure 44 shows the% decrease in platelet activation by different treatments. At concentrations as high as 50 pm, DS and SILY had little or no effect on the inhibition of platelet activation. Unlike individual DS or SILY, DS-SILY was able to inhibit collagen-mediated platelet activation. As the number of SILY peptides per DS molecule increased from 2 to 10, the inhibition of platelet activation also increased. Since the high SILY / DS ratio is expected to provide a higher affinity for collagen surface binding due to multiple interactions, peptidoglycans with the highest SILY / DS ratios were prepared.

[0132] The number of peptides per DS molecule was further increased to 18 to create the DS-SILY18 peptidoglycan, and the concentration of molecule necessary to inhibit platelet activation was tested. Figure 45 shows the degree of inhibition of platelet activation by DS-SILY18. The data together suggest that increasing the number of peptides per DS chain further inhibits platelet binding to collagen and platelet activation. Within the limits of solubility, the number of peptides can be increased to achieve maximum inhibition of platelets, as well as to reduce diffusion over time, where a higher level of peptoglycols is maintained in the wall of the bare vessel .

EXAMPLE 37

Diffusion of peptidoglycan from the collagen surface

[0133] DS-SILYi8.biotin peptidoglycan was dissolved at 10 pm in 1 x PBS pH 7.4 with 2% BSA and incubated in fibrillar collagen coated plates prepared as described. The plate was incubated at 37 ° C on an orbital shaker and rinsed thoroughly with 1xPBS pH 7.4. At several time points up to 11 days, DS-SILY18-biotin was detected on the surface as described for binding affinity studies. The peptidoglycan diffusion curve of the collagen surface was adjusted using a hyperbolic decay.

[0134] The diffusion of DS-SILY18 peptidoglycan from a collagen surface was measured by incubation at 37 ° C and rinsed extensively in order to mimic blood flow in vivo. Peptidoglycan was incubated on fibrillar collagen surfaces and detected by the same methods to calculate binding affinities. As shown in Figure 46, peptidoglycan diffuses to some extent from the surface of the collagen; however, after 1 week of extensive rinsing, the equivalent of approximately 10 nM remained attached to the surface. It is estimated that complete regrowth of endothelial cells occurs within a week after lesion of the balloon, and therefore this time frame is useful to prevent platelet binding until the endothelial cells grow back and provide a permanent cover to the underlying collagen.

EXAMPLE 38

Proliferation of endothelial cells

[0135] Endothelial regrowth is essential to restore the healthy vessel and provide a permanent barrier that covers the underlying collagen. The currently available drug-eluting stents, for example, prevent endothelial regrowth and, consequently, have demonstrated a new set of complications, such as late stent thrombosis. Peptidoglycan was tested at varying concentrations to determine if it inhibited endothelial regrowth. The peptidoglycan was physically bound to the collagen through peptide-collagen interactions, capable of being eliminated by competitive binding, and was thus replaced by endothelial cells that grew back on the collagen layer.

[0136] Endothelial cells (EC) of the human coronary artery (Lonza, Walkersville, MD) were seeded in 96-well plates at a cell density of 1.5x103 cells / well. The cells were allowed to adhere to the surface for 24 hours and the adherent ECs were stained using a green cell tracker (Invitrogen, Carlsbad, CA). The initial cell number was determined by measuring the fluorescence of each well with an excitation of 492 nm and an emission of 517 nm.

[0137] DS-SILY18 was solubilized in water at a concentration of 175 pm, and diluted in water 10 times for concentrations of 175, 17.5 and 1.75 pM. The DS-SILY solution was diluted 5 times with cell medium for final concentrations of 35, 3.5 and 0.35 mM. The control consisted of diluting water 5 times with cellular media. 100 µl of DS-SILY medium was added to each well and the cell number was determined 48 hours later by measuring the fluorescence. The percentage change in the number of cells in each well was calculated. As shown in Figure 47, at the highest concentration tested, there was a significant increase in cell proliferation, suggesting that peptidoglycan promotes endothelial regrowth instead of inhibiting regrowth.

EXAMPLE 39

Endothelial Cell Migration

[0138] To mimic the real environment where an area of endothelial cells developed in collagen is denounced, a second endothelial cell migration test was performed. Fibrillar collagen (Chronolog, Havertown, PA) was coated in wells of the 96-well Oris cell migration kit (Platypus Technologies, Madison, WI). Plugs were inserted into the plate to block an internal circular portion of the cell. Endothelial cells (EC) of the human coronary artery (Lonza, Walkersville, MD) were seeded at 5x103 cells / well and cultured to confluence outside the well. Once confluent in the outer portion of the well, the cells were stained with green cell tracking (Invitrogen, Carlsbad, CA). The plugs were removed from the wells and DSX ^SILY 18 solubilized in 1X PBS was incubated on the exposed collagen surface in the inner portion of the well for 15 minutes at 37 ° C. Unbound DS-SILY was rinsed from the surface and cell media was returned to the wells. The EC was allowed to migrate from the outside of the wells to the inner part for 48 hours. Fluorescence measurements from the center of each well were measured using a mask provided with the migration kit, so that only the treated inner circular portion of the well was measured.

[0139] As shown in Figure 48, the same trend of increasing the number of cells in increasing concentrations of peptidoglycan was observed. Along with cell proliferation trends, the data shows that endothelial regrowth is not inhibited by treatment with pepidoglycan, but that peptidoglycan promotes regrowth.

EXAMPLE 40

Platelet binding to collagen under flow

[0140] To mimic physiological conditions with flowing blood, platelet binding to collagen surfaces under flow conditions was evaluated. Flow kits were obtained from Ibidi (Munchen, Germany). Each channel was coated with fibrillar collagen (Chronolog). The excess collagen was removed from the flow channel by pushing IX PBS through the channel with a syringe. DS-SILY18 was incubated in the canal at a concentration of 50 pm for 15 min at 37 ° C, and the unbound peptidoglycan was rinsed by pushing IX PBS through the canal with a syringe. The control channel consisted of collagen not treated with peptidoglycan.

[0141] Platelet-rich plasma was propelled through the channels at 2 ml / h for 1 hour, which corresponds to a shear stress of 3.55 dynes / cm2 and a shear rate of 355 s-1. Unbound platelets were rinsed from the channel by pressing through IX PBS. The adhered platelets were fixed in the canal with 10% of formaldehyde. Platelets were permeabilized and actin filaments stained with phalloidin-Alexa Fluor 488 (Invitrogen). Representative images of adherent platelets are shown in Figure 49, which demonstrate that a significantly smaller number of platelets bind to the surface treated with peptidoglycan compared to untreated collagen.

EXAMPLE 41

In vivo studies of Ossabaw pig

[0142] In vivo studies were performed in Ossabaw pigs to determine the optimal method of administration and concentration of peptidoglycan, as well as to determine the preliminary efficacy of peptidoglycan treatment. Healthy adult Ossabaw pigs underwent PCI procedures following the protocols approved at the Indiana University School of Medicine. In these studies, a 10 mm balloon catheter was placed in several arteries with diameters of approximately 3 to 4 mm in diameter. The balloon was inflated to 1 to 1.3 times the diameter of the vessel to effectively strip the vessel, and was immediately followed by a Clearway® administration balloon sized to the vessel diameter.

[0143] Denuded arteries were treated by injection through the delivery balloon between 2 and 10 ml of either control saline solution or various concentrations of peptidoglycan dissolved in 1 x PBS pH 7.4. Contrast angiography was recorded before and after each treatment to monitor the position of the globe and the diameters of the vessels. After 14 days, the pigs were sacrificed and the vessels were collected for histological evaluation using H&E and Verhoff-Van Gieson staining. Pigs were heparinized during PCI procedures, but did not receive antiplatelet therapy at any time.

[0144] Denuted vessels treated with simulated control responded to balloon injury with significant vasospasm. Vasospasm is commonly observed in pigs and is a direct consequence of the binding and activation of platelets in the denoted endothelium. Vasospasm was used as a measure of the effective inhibition of platelet binding and the inhibition of platelet activation with peptidoglycan treatment. The severity of the vasospasm corresponds to the degree of platelet deposition in the denoted endothelium. The degree of vasospasm was quantified by measuring the diameter of the vessel before and after the balloon injury and treatment with ImageJ software, and the occlusion percentage was calculated.

[0145] As shown in Figure 51, the treatment of significantly inhibited vasospasm peptidoglycan and denoted endothelial binding platelets. At higher concentrations of 2.5 mg / ml or more, vasospasm is almost completely inhibited, which corresponds to in vitro studies in which this concentration shows a maximum inhibition of platelet activation.

EXAMPLE 42

Histological evaluation of vessels

[0146] Histological evaluation was performed in injured vessels by balloon 14 days after the injury to assess intimal hyperplasia. No adverse responses were observed, and preliminary results at high concentrations of peptidoglycan suggested that peptidoglycan inhibits intimal hyperplasia as shown in Figure 52.

Administration:

[0147] For optimal delivery of the peptidoglycan treatment, application on the denoted endothelium should occur immediately after the balloon injury. Peptidoglycan can prevent platelets from attaching to the denoted endothelium. A system in which a single balloon can be used capable of expanding the vessel and administering peptidoglycan treatment, however, effective platelet inhibition is achieved under current administration protocols.

EXAMPLE 43

Immunohistochemistry

[0148] Fresh carotid arteries were collected from pigs and placed in cold 1x PBS, and analyzed within 5 hours after collection. The arteries were opened and stripped with a rubber policeman and then cut into segments of approximately 4 mm and placed in a 96-well plate. The biotin-labeled DS-SILY18-biotin peptidoglycan was incubated at 10 pm dissolved in 1x PBS pH 7.4 for 15 min at room temperature. Control arteries were incubated with 1x PBS pH 7.4. The arteries were instantly frozen in liquid nitrogen, cut into sections of 7 pm and air dried for 45 min, then stored at -20 ° C until staining. The tissue was fixed in ice-cold acetone, air dried and washed with DI water. Sections were incubated with streptavidin-HRP for 30 minutes, washed with DI water, incubated with DAB for 10 minutes.

Claims (9)

1. A collagen binding peptidoglycan of formula (PnL) xG, in which n is from 1 to 7, x is from 1 to 10, P is a synthetic peptide of up to 40 amino acids comprising a sequence of RRANAALKAGELYKSILY, L is a linker, and G is glycan, For use in a method to treat: i) a vascular lesion; or ii) intimal hyperplasia. 2. The collagen binding peptidoglycan for use according to claim 1, wherein the compound is administered to a patient before, during or after vascular intervention. 3. The collagen binding peptidoglycan for use according to claim 2, wherein the vascular intervention is angioplasty, particularly balloon angioplasty. 4. The collagen binding peptidoglycan for use according to claim 1 or 2, wherein the collagen binding peptidoglycan is administered intravascularly. 5. The collagen binding peptidoglycan for use according to claim 1, wherein the linker has a molecular weight of 20 to 500 Daltons. 6. The collagen binding peptidoglycan for use according to claim 1, wherein the glycan is a glycosaminoglycan. 7. The collagen binding peptidoglycan for use according to claim 1, wherein the glycan is alginate, agarose, dextran, chondroitin, dermatan, dermatan sulfate, heparan, heparin, keratin or hyaluronan. 8. The collagen binding peptidoglycan for use according to claim 1, wherein the glycan is dermatan sulfate. 9. The collagen binding peptidoglycan for use according to claim 1, wherein the glycan is heparin.